Treatment of tissue by the application of energy

ABSTRACT

Methods and apparatuses for treating a tissue with an electric treatment by rotating a pattern of electrodes partway through a treatment is disclosed. Also described herein are methods and apparatuses to treat tissue, including treating skin disorders, by selectively de-nucleating epidermal cells without provoking a significant inflammatory response, e.g., without increasing the density of leukocytes in the treated skin, and without affecting the non-cellular components of the dermis.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 62/542,711, filed on Aug. 8, 2017 (titled “TREATMENT OFSKIN BY SELECTIVE ANUCLEATION OF EPIDERMAL CELLS”), herein incorporatedby reference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Specifically incorporate by reference in their entirety are each of:U.S. patent application Ser. No. 15/484,550, filed Apr. 11, 2017, U.S.patent application Ser. No. 13/631,618 filed Sep. 28, 2012 (now U.S.Pat. No. 9,656,055), and U.S. patent application Ser. No. 13/710,077,filed Dec. 12, 2011.

FIELD

This disclosure relates to treatment of tissue by the application ofpulsed electric fields, such as nanosecond electrical pulses. Thetreatment may selectively and specifically destroy the nuclei of treatedcells (e.g., epithelial cells) without provoking a significantinflammatory response, and while sparing the adjacent non-cellulartissue.

BACKGROUND

The application of destructive modalities for the treatment of tissue iswell known. For example, many skin treatments, including treatment ofskin disorders, by the application of thermal modalities is well knownin dermatology. Thermal treatments in particular, including the use ofliquid nitrogen (e.g., −196° to −210° C.) to treat or remove affectedskin, are well known, but may result in severe disruption and immediatenecrosis of skin cells and bursting of the cell membrane, leading to anacute inflammation response, loss of melanocytes, and damage to thedermis, that can result in scar tissue formation and an abnormalappearance.

Other thermal treatment modalities that result in tissue destructioninclude tissue heating generated by laser or radio frequency deviceswhich may effectively burn the tissue (including skin) and may causeimmediate cell necrosis and destruction of cell membranes and may alsoprovoke an inflammatory response and suffer from the same drawbacks asextreme cold. It would be beneficial to provide therapies, and inparticular, non-thermal therapies, which produce a minimal, if any,local inflammatory response. As applied to dermal tissue, it would beparticularly helpful to provide for the formation of new epidermaltissue with reduced or no significant scarring and a normal appearanceafter restoration of the epidermal surface after a normal healingperiod.

Ultra-short, high-field strength electric pulses have been described forelectroperturbation of biological cells. For example, electric pulsesmay be used in treatment of human cells and tissue including tumorcells, such as basal cell carcinoma, squamous cell carcinoma, andmelanoma. See, e.g., Garon et al. “In Vitro and In Vivo Evaluation and aCase Report of Intense Nanosecond Pulsed Electric Field as a LocalTherapy for Human Malignancies”, Int. J. Cancer, vol. 121, 2007, pages675-682, incorporated herein by reference it its entirety.

The voltage induced across a cell membrane may depend on the pulselength and pulse amplitude. Pulses longer than about 1 microsecond maycharge the outer cell membrane and lead to opening of pores. Permanentopenings may result in instant or near instant cell death. Pulsesshorter than about 1 microsecond may affect the cell interior withoutadversely or permanently affecting the outer cell membrane and result ina delayed cell death with intact cell membranes. Such shorter pulseswith a field strength varying, for example, in the range of 10 kV/cm to100 kV/cm may trigger apoptosis (i.e. programmed cell death) in some orall of the cells exposed to the described field strength and pulseduration. These higher electric field strengths and shorter electricpulses may be useful in manipulating intracellular structures, such asnuclei and mitochondria.

Nanosecond high voltage pulse generators have been proposed forbiological and medical applications. For example, see: Gundersen et al.“Nanosecond Pulse Generator Using a Fast Recovery Diode”, IEEE 26thPower Modulator Conference, 2004, pages 603-606; Tang et al.“Solid-State High Voltage Nanosecond Pulse Generator,” IEEE Pulsed PowerConference, 2005, pages 1199-1202; Tang et al. “Diode Opening SwitchBased Nanosecond High Voltage Pulse Generators for Biological andMedical Applications”, IEEE Transactions on Dielectrics and ElectricalInsulation, Vol. 14, No. 4, 2007, pages 878-883; Yampolsky et al.,“Repetitive Power Pulse Generator With Fast Rising Pulse” U.S. Pat. No.6,831,377; Schoenbach et al. “Method and Apparatus for IntracellularElectro-Manipulation”, U.S. Pat. No. 6,326,177; Gundersen et al.,“Method for Intracellular Modifications Within Living Cells Using PulsedElectric Fields”, U.S. Patent Application No. 2006/0062074; Kuthi etal., “High Voltage Nanosecond Pulse Generator Using Fast Recovery Diodesfor Cell Electro-Manipulation”, U.S. Pat. No. 7,767,433; Krishnaswamy etal., “Compact Subnanosecond High Voltage Pulse Generation System forCell Electro-Manipulation”, U.S. Patent Application No. 2008/0231337;and Sanders et al. “Nanosecond Pulse Generator”, U.S. Patent ApplicationNo. 2010/0038971. The entire content of these publications isincorporated herein by reference.

Described herein are methods and apparatuses for the treatment oftissues, including skin, an in particular the treatments of skindisorders, which may address the issues raised above. These methods andapparatuses may target the nuclei of epidermal cells specifically,including the use of ultra-short, high field strength electric pulses.

SUMMARY OF THE DISCLOSURE

The methods, systems and apparatuses described herein generally describethe application of electric energy treatment(s) to tissue to form alesion in the tissue while permitting healing. Skin tissue in particularis described as a possible target of the methods and apparatusesdescribed herein, however it should be noted that these methods andapparatuses are not limited to the treatment of skin.

For example, in some variations, described herein are methods andapparatuses for applying a treatment to a tissue from a plurality ofelectrodes by dividing the treatment (e.g., treatment dose) into two ormore parts, and rotating the applicator tip so that the electrodes applythe energy to the same portion of tissue from multiple differentrotational orientations during the treatment. The inventors havesurprisingly found that rotation of the applicator tip part way throughthe treatment (e.g., approximately halfway through the treatment)requires substantially fewer pulses, e.g., less energy, in order to getan equivalent treatment compared to treatments in which the applicatortip is not rotated, and it provides further benefits as disclosedherein. For example, rotating the treatment tip having a pattern ofelectrodes by 90 degrees during treatment results in similar sizedtreatment-induced lesions using fewer treatment pulses compared totreatments applied with more pulses but without rotating electrodes.

Thus, described herein are methods and apparatuses for rotating thetreatment electrodes partway through a treatment. For example, describedherein are methods of treating a tissue by applying pulsed electricalenergy (in some examples comprising a plurality of nanosecond electricalpulses having a pulse duration of between 0.1 ns and 1000 ns), whereinthe treatment is divided into a first portion and a second portion. Insome implementations, the method may include: contacting the tissue withan applicator tip (e.g., treatment tip) having a pattern of electrodes;applying the first portion of the treatment to a region of the tissuethrough the pattern of electrodes with the pattern of electrodescontacting the region of the tissue in a first orientation; and applyingthe second portion of the treatment to the region of the tissue in thesame pattern of electrodes a second orientation that is rotated relativeto the first orientation. The rotation may be about a line of rotationthrough the plurality of electrodes (e.g., a midline through thetreatment tip and/or though the plurality of electrodes). The sameelectrodes of the treatment tip may form the pattern of electrodesapplied in the first orientation as in the second orientation; also thepattern may be formed by all of the electrodes of the treatment tip.

The treatment tip (and therefore the pattern of electrodes) may berotated any appropriate amount, including, e.g., between +/−1 degree and359 degrees, between +/−1 degree and 179 degrees, between +/−5 degreesand +/−175 degrees, between +/−70 degrees and 110 degrees, etc. Forexample, applying the second portion may comprise applying the secondportion of the pulsed electrical treatment through the electrodes in thesecond orientation that is rotated between 80 degrees and 100 degreesrelative to the first orientation. Applying the second portion maycomprise applying the second portion of the pulsed electrical treatmentthrough the electrodes in the second orientation that is rotated 90degrees relative to the first orientation. Also, a degree or amount ofrotation from the first orientation to the second orientation may beuser selected (for example, through a user interface) or automaticallydirected by a controller.

Generally, the treatment before and after rotation is applied to thesame region of the tissue. For example, applying the second portion maycomprise applying the second portion so that the second orientationoverlaps with the first orientation on the first region of the tissue.

The treatment may be divided up into any number of portions (e.g., 2, 3,4, 5, 6, etc.). In some variations the tissue may be divided up into twoportions. In general, the first portion of the pulsed electricaltreatment may be between 30% and 70% of the total treatment (e.g.,treatment dose). For example, the first portion of the treatment may bebetween 40% and 60% of the pulsed electrical treatment. The firstportion of the pulsed electrical treatment may be half of the treatment.

Any appropriate type of electrode may be used, including penetratingelectrodes (e.g., needle electrodes, blade electrodes, etc.) ornon-penetrating electrodes (e.g., surface electrodes). In somevariations the treatment tip includes an array of needle electrodes. Theelectrodes may be fixed relative to the distal face of the treatmenttip, or they may be configured to retract relative to the treatment tip(e.g., retract into the treatment tip).

Rotation of the electrodes (e.g., including rotation of the treatmenttip holding the pattern of electrodes) may include removing thetreatment tip and/or electrodes from the tissue before rotation. Forexample, the method may include removing the plurality of electrodesfrom the region of the tissue, rotating the applicator tip andre-applying the plurality of electrodes to the region of the tissuebefore applying the second portion of the pulsed electrical treatment.

In any of the methods described herein, the rotation of the electrodesmay be performed manually, semi-manually, or automatically. The rotationof the electrodes (e.g., rotation of the applicator, applicator tipand/or the pattern of electrodes) between different portions of thetreatment may be performed robotically. In addition, any or all of thesteps of the methods disclosed herein, including removing, rotating andreapplying the electrodes, as well as coordinating application of thepulsed electrical energy, may be performed by a robotic system, forexample, under computer control.

According to another aspect, in some examples described herein, insteadof rotating the applicator tip, a pattern of active electrodes may berotated to achieve the benefits described above. In some variations thepattern of electrodes applying energy to the same region of tissue maybe rotated by switching the electrodes (either mechanically and/orelectrically). For example, the pattern of electrodes on the tip of theapplicator may be formed from a sub-set of electrodes available on thetreatment tip. The apparatus may electrically and/or mechanically switchwhich electrodes form the pattern, and therefore, the orientation of thepattern on the applicator tip. Thus, the methods may include rotatingthe pattern of electrodes of the applicator tip on or in the region ofthe tissue without removing the applicator tip from the region of thetissue. For example, the tissue may be contacted during treatment withan applicator tip having an array of electrodes in which the pattern ofelectrodes is formed of a first subset of active electrodes from thearray of electrodes; further wherein applying the second portion of thepulsed electrical treatment may include forming the pattern from asecond subset of active electrodes from the array of electrodes in whichthe pattern formed by the second subset is rotated relative to the firstsubset. In some examples, the method of treating a tissue with pulsedelectrical energy comprising a plurality of electrical pulses isprovided. The method comprises applying pulsed electrical energy from asubset of electrodes of an array of electrodes in a first pattern; andpartway through the pulsed electrical treatment switching to a secondsubset of electrodes of the array of electrodes to apply electricalenergy in a second pattern that is a rotated version of the firstpattern. The switching may be electrical, e.g., switching whichelectrodes are active (applying energy) and which are not active. Insome variations the switching may be mechanical, e.g., changing themechanical connection of the different electrodes applying power. Insome variations the electrodes may be switched by moving electrodes intoor out of the tissue.

In general, applying the pulsed electrical treatment may includeapplying a pulsed electrical treatment that does not disrupt cellmembranes within the tissue.

The treatment may include applying a plurality of pulses each having aduration of between 0.1 ns and 1000 ns and. For example, applying thefirst portion and/or second portion of the pulsed electrical treatmentcomprises applying a plurality of pulses each having a duration ofbetween 0.1 ns and 1000 ns and a peak field strength of at least 1kV/cm.

According to one example of general methodology, described herein is amethod of treating a tissue with a pulsed electrical treatmentcomprising a plurality of electrical pulses, wherein the pulsedelectrical treatment is divided into at least a first portion and asecond portion. The method comprises: contacting the tissue with anapplicator tip having a plurality of electrodes; applying the firstportion of the pulsed electrical treatment to a region of the tissue ina pattern of electrodes from the plurality of electrodes, the pattern ofelectrodes contacting the region of the tissue in a first orientation;and applying the second portion of the pulsed electrical treatment tothe region of the tissue in the pattern of electrodes contacting theregion of the tissue in a second orientation that is rotated relative tothe first orientation. The pattern of electrodes in the secondorientation may be formed by the same electrodes as the firstorientation or some or all of them may be different. Also, the patternmay be formed by all of the electrodes of the plurality or electrodes,or only by a portion of the plurality of electrodes. For example, insome examples, the second portion of the pulsed electrical treatment isapplied through the same plurality of electrodes. In other examples, theapplicator tip comprises an array of electrodes in which the pluralityof electrodes is a first subset of active electrodes forming the patternof electrodes and applying the second portion of the pulsed electrictreatment comprises forming the pattern from a second subset of activeelectrodes from the array of electrodes in which the pattern formed bythe second subset is rotated relative to the first subset. The treatmentin any of the above methods may be any appropriate duration. Forexample, the treatment may be between 10 seconds and 20 minutes, between10 seconds and 10 minutes, between 10 second and 5 minutes, less than 10minutes, less than 7 minutes, less than 5 minutes, less than 4 minutes,etc. For example, applying the first and second portions of the pulsedelectrical treatment may comprise applying for less than 5 minutes.

The second orientation may be rotated about a midline through theplurality of electrodes.

As mentioned, any appropriate tissue, e.g., skin, liver, kidney, lung,etc. including tumor (e.g., tumorous tissue) is within a scope of thepresent disclosure. For example, contacting the tissue may comprisecontacting a skin tissue. Any of these methods may be methods oftreating the tissue, including methods of selectively removing tissue.For example, the skin tissue treated may comprise one or more of:seborrheic keratosis, keloids, molluscum contagiosum, sebaceoushyperplasia, syringoma, congenital capillary malformation (port-winestain), melasma, actinic keratoses, dermatosis papulosa nigra,angiofibroma, skin tumors, and warts. Contacting may comprise contactinga tumor tissue.

As will be described in greater detail below, applying the pulsedelectrical treatment may increase a marker of inflammation within thefirst region of the tissue by less than 15%, wherein the marker ofinflammation is one or of more of: fibroblast density, leukocytedensity, Interleukin-6, Interleukin-8, Interleukin-18, Tumor necrosisfactor-alpha, and C-reactive protein.

According to further examples, described herein are methods of treatinga tissue with a pulsed electrical treatment comprising a plurality ofnanosecond electrical pulses having a pulse duration of between 0.1 nsand 1000 ns, wherein the treatment is divided into a first portion and asecond portion. The first portion may be between 30% and 70% of thepulsed electrical treatment (e.g., treatment duration). The method mayinclude: contacting the tissue with an applicator tip having a pluralityof electrodes in a pattern of electrodes; applying the first portion ofthe pulsed electrical treatment to a region of the tissue through theplurality of electrodes with the pattern of electrodes contacting theregion of the tissue in a first orientation; removing the plurality ofelectrodes from the region of the tissue; rotating the applicator tip;re-applying the plurality of electrodes to the region of the tissue; andapplying the second portion of the pulsed electrical treatment to theregion of the tissue through the plurality of electrodes with thepattern of electrodes contacting the region of the tissue in a secondorientation that is rotated relative to the first orientation.

A further method of treating a tissue with a pulsed electricaltreatment, wherein the pulsed electrical treatment is divided into afirst portion and a second portion, may include: contacting a region ofthe tissue with an applicator tip having a plurality of electrodes in apattern of electrodes; applying the first portion of the pulsedelectrical treatment to the region of the tissue through the pluralityof electrodes with the pattern of electrodes contacting the region ofthe tissue in a first orientation; removing the plurality of electrodesfrom the region of the tissue; re-applying the plurality of electrodesto the region of the tissue with the pattern of electrodes contactingthe region of the tissue in a second orientation that is rotatedrelative to the first orientation; and applying the second portion ofthe pulsed electrical treatment to the first region of the tissuethrough the plurality of electrodes. In some variations, the secondorientation may be rotated between 40 degrees and 100 degrees relativeto the first orientation, for example, 90 degrees. According to someembodiments, applying the first portion of the pulsed electricaltreatment and applying the second portion of the electrical treatmenteach comprises applying electrical pulses to the region to de-nucleatecells within the region without provoking a substantial inflammatoryresponse, so that after the treatment the tissue forms a necrotic crustover the region so that when the necrotic crust is removed new tissue isexposed.

Also described herein are apparatuses (e.g., systems and devices)configured to perform any of these methods, including methods oftreating the tissue with pulsed electrical energy and rotating thepattern of electrodes partway through the treatment. For example, asystem may include a pulse generator; an applicator having a pluralityof electrodes at a treatment tip of the applicator, the applicator tipconfigured to apply energy from the pulse generator to the plurality ofelectrodes; and a controller configured to control, at least partially,operation of the pulse generator and the applicator tip. The controllermay comprise a processor having a set of instructions, wherein the setof instructions, when executed by the processor causes the controller toapply a first portion of the pulsed electrical treatment in a pattern ofelectrodes from the plurality of electrodes in a first orientation andapply a second portion of the pulsed electrical treatment in the patternof electrodes in a second orientation that is rotated relative to thefirst orientation. In some implementations, the set of instructionscomprises instructions for applying the second portion of the pulsedelectrical treatment through the same plurality of electrodes andwherein the second orientation is rotated about a midline through theplurality of electrodes. In other implementations, the applicator tipcomprises an array of electrodes in which the plurality of electrodes isa first subset of active electrodes forming the pattern of electrodesand the set of instructions comprises instructions wherein applying thesecond portion of the pulsed electric treatment comprises forming thepattern from a second subset of active electrodes from the array ofelectrodes in which the pattern formed by the second subset is rotatedrelative to the first subset.

According to some examples a system for treating tissue may include: apulse generator; an applicator configured to apply energy from the pulsegenerator to a plurality of electrodes at a treatment tip of theapplicator, wherein the plurality of electrodes is arranged about a lineof rotation through the treatment tip; and a controller configured tocontrol, at least partially, operation of the pulse generator and theapplicator. The controller comprises a processor having a set ofinstructions, wherein the set of instructions, when executed by theprocessor, causes the controller to: apply a first portion of the pulsedelectrical treatment from a first pattern of electrodes of the pluralityof electrodes at the treatment tip; and apply a second portion of thepulsed electrical treatment through the plurality of electrodes in asecond pattern of electrodes wherein the second pattern of electrodes isthe first pattern of electrodes rotated about the line of rotation.

The set of instructions may further cause the processor and/orcontroller to rotate the treatment tip to form a second pattern ofelectrodes of the plurality of electrodes at the treatment tip. In somevariations, the set of instructions further causes the processor toswitch electrical or mechanical connections of at least some of theelectrodes in the plurality of electrodes to form the second pattern ofelectrodes of the plurality of electrodes at the treatment tip.

The line of rotation may be a midline through the plurality ofelectrodes.

The pulse generator may be configured to deliver pulses having a pulseduration of between 0.1 ns and 1000 ns. In some variations, the pulsegenerator is configured to deliver a plurality of pulses each having aduration of less than 1 microsecond and a peak field strength of atleast 1 kV/cm.

Any of these apparatuses may include an actuator (e.g., motor, driver,impeller, etc.) configured to rotate an applicator or at least a portionof the distal tip of the applicator under the control of the controllerand/or processor.

The system may include an electrical switching module configured toswitch between the first pattern and the second pattern of theelectrodes, for example, between a first and a second subset of activeelectrodes. Alternatively or additionally, the system may includemechanical switches for controlling which electrodes are active orinactive.

The second pattern may be identical to the first pattern but rotated anyamount (e.g., between 40 degrees and 100 degrees, between 20 and 180degrees, between 30 and 80 degrees, etc.) relative to the first pattern.The degree or amount of rotation from the first orientation to thesecond orientation may be user selected (for example, through a userinterface) or automatically directed by a controller, for example,through the set of instructions.

Any of these systems may be robotic systems wherein the applicatorcomprising a treatment tip with an array of electrodes is coupled to amoveable arm. For example, the robotic system may receive instructionsfrom the controller and rotate one or both of the applicator and thetreatment tip to change orientation of the pattern or electrodes.

For example, a system for applying pulsed electrical treatment to atissue may include: a movable arm (e.g., robotic arm); an applicatoroperatively coupled to the movable arm, the applicator configured toapply pulsed electrical energy from a plurality of electrodes of theapplicator; and a processor comprising a set of instructions forexecuting operations, the set of instructions including instructionsfor: moving the movable arm to contact a region of the tissue with theapplicator; directing application of a first portion of the pulsedelectrical treatment to the region of the tissue with a pattern ofelectrodes contacting the region of the tissue in a first orientation;and directing application of a second portion of the pulsed electricaltreatment to the region of the tissue with the pattern of electrodescontacting the region of the tissue in a second orientation that isrotated relative to the first orientation.

In some examples, the robotic system may include a navigation interfacecomprising, for example, an image acquisition device and the navigationinterface may be configured to receive imaging data. In general, thenavigation interface may determine the distance between the tissue (aswell as the location of the target treatment site on the tissue) and theplurality of electrodes/treatment tip, and/or the orientation of theplurality of electrodes/treatment tip and the tissue, to allow controland guidance of the treatment tip relative to the tissue.

The applicator may be operably connected to the movable arm, such asheld by the movable (e.g., robotic) arm. Alternatively, the applicatormay be integrated into the movable arm.

The set of instructions may comprise instructions for moving theapplicator so that that the plurality of electrodes is moved to thesecond orientation. For example, the set of instructions may compriseinstructions for withdrawing the applicator from the tissue, rotatingthe applicator so that that the plurality of electrodes is moved to thesecond orientation, and reapplying the applicator tip to the region ofthe patient tissue.

As mentioned, any of the methods and apparatuses (e.g., devices andsystems, including applicators) described herein may be used to treatskin, including treating skin disorders, including but not limited toseborrheic keratosis, keloids, molluscum contagiosum, acrocordon,psoriasis, papilloma, human papilloma virus (HPV), melanoma, melasma,sebaceous hyperplasia, syringoma, congenital capillary malformation(port-wine stains), melasma, actinic keratosis, dermatosis papulosanigra, angiofibroma, skin tumors, aged skin, wrinkled skin, and warts.These methods and apparatuses may also be used for cosmetic skintreatments, including tattoo removal, hair follicle destruction, scarreduction and wrinkle reduction.

Also described herein are methods and apparatuses for treating a skinlesion of a mammal (including human and non-human mammals) that maygenerally include the application of pulsed electrical energy to adefined region of a patient's skin in which pattern of electrodesapplying the treatment to the defined region are rotated partway throughthe treatment. In general, these treatments make targeted cells withinthe epidermal or dermal region non-viable, typically within 2-48 hoursfollowing treatment. For example, the methods described herein mayde-nucleate cells (and particularly epidermal cells), forming “ghostcells” that may have intact cell membranes, but may lack a distinct cellnucleus. Furthermore, the application of the treatment may be configured(e.g., titrated, limited, arranged, etc.) so that the de-nucleation doesnot result in a substantial inflammatory response. For example, in skin,as a result of the targeted de-nucleation of the epidermal cells in thetarget region, the skin in that region may form a necrotic crust, andnew epidermis may form below this necrotic crust so that when thenecrotic crust is removed, the new epidermis is exposed. Rotating thepattern of the electrodes applying the energy to the skin in the targetregion may result a higher efficiency treatment, as less energy may beneeded to treat an equivalent-sized (or larger) region. This newepidermis may include epidermal cells (e.g., newly formed epidermalcells) that have healthy nuclei, including a normal (as compared toother adjacent skin regions) distribution of melanocytes, and normalelastin distribution and density. Thus, unlike other methods of treatingskin, e.g., to remove skin lesions, the resulting skin region may havelittle or no scarring and/or discoloration.

In some variations, the pattern of electrodes applying the electricalenergy to a target skin region is not rotated during treatment. Forexample, also described herein are methods of treatment of a skin lesionof a mammal (including human and non-human mammals) that may generallyinclude the application of pulsed electrical energy to a defined regionof a patient's skin. In general, these treatments make targeted cellswithin the epidermal or dermal region non-viable, typically within 2-48hours following treatment. The methods described herein may de-nucleatecells (and particularly epidermal cells), forming ghost cells. Theapplication of the treatment may be configured (e.g., titrated, limited,arranged, etc.) so that the de-nucleation does not result in asubstantial inflammatory response. For example, in skin, as a result ofthe targeted de-nucleation of the epidermal cells, the skin in theregion may form a necrotic crust, and new epidermis may form below thisnecrotic crust so that when the necrotic crust is removed, the newepidermis is exposed. This new epidermis may include epidermal cells(e.g., newly formed epidermal cells) that have healthy nuclei, includinga normal (as compared to other adjacent skin regions) distribution ofmelanocytes, and normal elastin distribution and density, with little orno scarring and/or discoloration.

For example, described herein are methods of treating a skin disorder,the methods including: applying a pulsed electrical treatment to aregion of the skin to de-nucleate epidermal cells within the regionwithout provoking a substantial inflammatory response, so that the skinforms a necrotic crust over the region of the skin and forms newepidermis below the necrotic crust so that when the necrotic crust isremoved the new epidermis is exposed. The methods may comprise restoringelastin integrity of the skin. Any of these methods of treating a skindisorder may include the steps of rotating the pattern of the electrodesapplying the pulsed electrical treatment partway through the applying ofthe treatment. This may include diving the treatment up into a firstpart and a second part and rotating the pattern of electrodes used inthe first part of the treatment and applying the rotated pattern to thesame region of the skin during the second part of the treatment.

Any appropriate pulsed electrical treatment may be used. In somevariations, a treatment that specifically de-nucleates epidermal cellswithout provoking a substantial inflammatory response may be used. Inparticular, the treatment may include the use of nano-pulse stimulation(NPS), e.g., the application of nanosecond electrical pulses having apulse duration of between 0.1 ns and 1000 ns and a high field density,e.g., a field strength of at least 1 kV/cm or greater to the epidermalcells or other tissue cells. However, other treatments for specificallyde-nucleating epidermal cells may include optical therapies (e.g., theuse of photosensitive nuclear stains in conjunction with opticalstimulation), and the like. Generally, the treatment according to thepresent disclosure may include any treatment that does not disrupt thecell membrane of the epidermal cells.

For example, applying the treatment may include inserting a pair ofelectrodes (or a plurality of electrodes) into the region of the skinand applying a plurality of high voltage nanosecond electrical pulsesbetween the electrodes. Alternatively or additionally, applying thetreatment may comprise applying a pair of electrodes against the regionof the skin (e.g., non-invasively) and applying a plurality of highvoltage nanosecond electrical pulses from the electrodes. Anon-conductive gel or other material may be used, and/or may beintegrated into the electrodes and/or applicator.

As mentioned, applying the treatment (e.g., pulsed electrical energytreatment) may comprise applying a plurality of pulses each having aduration of between 0.1 ns and 1000 ns and a peak field strength of atleast 1 kV/cm. Applying the treatment may include applying the treatmentfor less than 10 minutes (e.g., less than 1 second, less than 2 seconds,less than 5 seconds, less than 10 seconds, less than 15 seconds, lessthan 30 seconds, less than 45 seconds, less than 1 minute, less than 2minutes, less than 3 minutes, less than 4 minutes, less than 5 minutes,etc.).

In general, applying the treatment does not provoke a substantialincrease in inflammation. For example, applying the treatment mayincrease a marker of inflammation within the region of the skin by lessthan a predefined percentage (e.g., 5% or less, 10% or less, 15% orless, 20% or less, etc.). The marker of inflammation may be one or ofmore of: leukocyte density, Interleukin-6, Interleukin-8,Interleukin-18, Tumor necrosis factor-alpha, and C-reactive protein. Inparticular, a marker of acute inflammation such as the leukocytedensity, may be increased by less than a predefined percentage (e.g.,15%) compared prior to treatment.

As mentioned above, the methods and apparatuses described herein may beused to treat a skin disorder, including but not limited to one or moreof: seborrheic keratosis, keloids, molluscum contagiosum, sebaceoushyperplasia, syringoma, congenital capillary malformation (port-winestains), melasma, actinic keratosis, dermatosis papulosa nigra,angiofibroma, skin tumors, and warts. Any of these methods may also beused to treat otherwise healthy skin including cosmetic blemishes, suchas tattoos, wrinkles, and scars.

For example, described herein are methods of treating a patient's skin,the method comprising: positioning a set of electrodes in communicationwith a region of a patient's skin; applying a plurality of high-fieldstrength, ultra-short electrical pulses to the region from the set ofelectrodes to de-nucleate epidermal cells, such that the skin forms anecrotic crust over the region of the skin and forms new epidermis belowthe necrotic crust so that when the necrotic crust is removed the newepidermis is exposed; wherein the high-field strength, ultra-shortelectrical pulses comprise a plurality of pulses each having a durationof between 0.1 ns and 1000 ns and a peak field strength of at least 1kV/cm.

Any of these methods may include inserting a pair of electrodes into thepatient's skin before applying the plurality of high-field strength,ultra-short electrical pulses. For example, the electrodes may beinserted into the outer layers of skin to a depth of less than 5 mm,less than 4 mm, less than 3 mm, less than 2 mm, etc. The skin may beprepared ahead of time, e.g., washed, shaved, roughened, etc.Alternatively or additionally, the high-field strength, ultra-shortelectrical pulses may be applied transdermally, without puncturing theskin. For example, any of these methods may include applying the set ofelectrodes on the surface of the patient's skin before applying theplurality of high-field strength, ultra-short electrical pulses. In suchvariations one or more conductive or non-conductive gels or othermaterials may be applied to the skin, including to the electrode contactpoints and/or the region between them. For example, a non-conductive orlower-conductance gel may be used. Alternatively or additionally, a gel(low-conductance or non-conductive gels) may be used with needleelectrodes.

While in some variations a pair of electrodes may be used, in othervariations more than two electrodes (e.g., two or more active electrodesand two or more ground electrodes) may be used. The active electrodesmay be coupled together; the ground electrodes may be coupled together.

Applying the plurality of high-field strength, ultra-short electricalpulses may include applying the high-field strength, ultra-shortelectrical pulses for less than a predetermined time (e.g., 1 second orless, 2 seconds or less, 5 seconds or less, 10 seconds or less, 15seconds or less, 30 seconds or less, 45 seconds or less, 1 minute orless, 2 minutes or less, 3 minutes or less, 4 minutes or less, 5 minutesor less, 10 minutes or less, 15 minutes or less, etc.) and/or for apredetermined number of pulses (e.g., between 2 and 30 pulses, between 2and 60 pulses, between 2 and 120 pulses, between 2 and 240 pulses,between 2 and 680 pulses, etc.). The pulses may be applied at anyappropriate frequency. For example, the plurality of high-fieldstrength, ultra-short electrical pulses may be applied between 0.05 Hzand 100 Hz (e.g., between 0.05 Hz and 20 Hz, between 0.05 Hz and 10 Hz,etc.).

As mentioned above, applying the plurality of high-field strength,ultra-short electrical pulses may increase a marker of inflammationwithin the region of the skin by less than a predetermined amount (e.g.,less than 5%, less than 10%, less than 15%, etc.), wherein the marker ofinflammation is one or of more of: leukocyte density, Interleukin-6,Interleukin-8, Interleukin-18, Tumor necrosis factor-alpha, andC-reactive protein. In particular, the marker may be an acuteinflammatory marker, such as (but not limited to) leukocyte density.

The step of positioning the pair of electrodes may comprise positioningthe pair of electrodes in communication with a region of skin having oneor more of: a seborrheic keratosis, a keloid, a molluscum contagiosum, asebaceous hyperplasia, a syringoma, a congenital capillary malformation(port-wine stain), a melasma, an actinic keratosis, a dermatosispapulosa nigra, an angiofibroma, a wart, and a tattoo.

In general, when applying nano-pulse stimulation (NPS), the electricalenergy applied to the skin lesion may be in the form of one or moreelectrical pulses. The pulse duration may be at least 0.01 nanoseconds(ns) at the full-width-half-maximum (FWHM). The pulse duration may alsobe at least 1 ns at FWHM, or the pulse duration may be at least 5 ns atFWHM. The pulse duration may be 1,000 ns or shorter.

The methods and apparatuses may be used to treat skin, includingtreating a skin lesion. As used herein, a skin lesion may refer to anydeviation of skin from a healthy or a normal condition. Examples of skinlesions are skin diseases, conditions, injuries, defects, abnormalitiesor combinations thereof. For example, such skin lesions includemalignancies (such as basal cell carcinomas, squamous cell carcinomasand melanoma), precancerous lesions (such as actinic keratosis), humanpapilloma virus (HPV) infected cells (such as verruca vulgaris or commonwarts, plantar warts, genital warts), immune-related conditions (such aspsoriasis), other skin abnormalities (such as seborrheic keratosis andacrocordon), or combinations thereof. In one embodiment, the skin lesionis basal cell carcinoma (including papilloma), squamous cell carcinoma,actinic keratosis, warts, or combinations thereof. The skin lesion mayalso include aged skin, wrinkled skin or damaged skin. An example of thedamaged skin is the skin damaged by sun radiation.

As mentioned, the duration of the pulse may be in the range of 0.01 nsto 1,000 ns. The duration of the pulse may also be in the range of 1 nsto 600 ns (e.g., 10 ns to 500 ns, 10 ns to 400 ns, etc.). In someimplementations, the duration of the pulses may be in a picosecondranges, or microsecond ranges, just to name a few. The appliedelectrical energy per volume of the skin lesion may be at least 10mJ/mm³, or at least 100 mJ/mm³, or at least 1,000 mJ/mm³. The appliedelectrical energy per volume of the skin lesion may also be in the rangeof 0.1 mJ/mm³ to 10,000 mJ/mm³.

The electrical field produced by each pulse may be at least 1 kV/cm atthe peak amplitude of the pulse. The electrical field produced by eachpulse may also be at least 10 kV/cm at the peak amplitude of the pulse.The electrical field produced by each pulse may be in the range of 1kV/cm to 1,000 kV/cm at the peak amplitude of the pulse (e.g., theelectrical field produced by each pulse may be in the range of 10 kV/cmto 100 kV/cm, 15 kV/cm to 50 kV/cm, 20 kV/cm to 30 kV/cm, etc.).

The number of electrical pulses during a single treatment may be atleast 1. The number of pulses may also be at least 100. The number ofpulses may be at least 1,000. The number of pulses may be less than10,000. For example, the number of pulses may be between 20 and 200,between 30 and 150, between 30 and 100, etc. Pulses may be applied at afrequency of between 1 and 100 Hz, e.g., between 1 and 50 Hz, between 1and 25 Hz, between 1 and 20 Hz, between 1 and 10 Hz, between 2 and 6 Hz,etc. The treatment time per session may be between 1 second and 60seconds, between 5 seconds and 30 seconds, between 5 seconds and 20seconds, etc.

The treatment may be an in vivo treatment of a skin lesion of a humancomprising at least one treatment session, i.e. administration of theelectrical energy to the skin lesion by physician at an office visit.The at least one treatment session may comprise applying electricalenergy to the skin lesion of the human comprising delivering at leastone electrical pulse with a pulse duration in the range of 0.01 ns to1,000 ns, forming an electrical field in the lesion, and thereby atleast preventing growth of the lesion. This pulse duration may also bein the range of 1 ns to 600 ns (e.g., 1 ns to 300 ns, 1 ns to 200 ns,etc.).

The treatment of a lesion may also comprise a plurality of treatmentsessions. For example, it may comprise at least two treatment sessionsor at least three treatment sessions. As used herein, the term skinlesion may also be referred to as a skin disorder, and/or lesions may beformed on or in the skin as part of a skin disorder. The methodsdescribed herein may be used to treat either the skin disorder and/or alesion of a skin disorder.

Also described herein are apparatuses (e.g., systems and devices) forperforming any of these methods, including the methods of treating askin disorder. For example, according to one aspect of the presentdisclosure a system for treating tissue is provided. The systemcomprises a pulse generator; a set of electrodes; and a controllerconfigured to control, at least partially, operation of the pulsegenerator, the controller comprising a processor having a set ofinstructions, wherein the set of instructions, when executed by theprocessor causes the pulse generator to generate and apply through theset of electrodes a pulsed electrical treatment to a region of tissue tode-nucleate cells within the region without provoking a substantialinflammatory response, so that the tissue forms a necrotic crust andforms new tissue below the necrotic crust so that when the necroticcrust is removed the new tissue is exposed.

The system for treating tissue may be a system for treating a skindisorder or skin lesion. Thus, the system used for the treatment of theskin lesion may include an applicator tip that comprises at least onedelivery electrode and at least one ground electrode. The applicator(e.g., applicator tip) may be any of the applicator tips describedherein, including arrays of electrodes and/or applicator tips having apattern of electrodes that may be rotated.

The pulse generator may be configured to provide pulses (including butnot limited to nanopulses) to be delivered by the applicator. The pulsegenerator and/or tip may be controlled by the controller. The controllermay include one or more processors that may be configured to perform anyof the treatment methods described herein. The one or more processorsmay be incorporated into the controller or may be a separate part. Thecontroller and/or processor may include one or more memories,datastores, or the like that may be operationally connected to theprocessor(s).

The set of instructions executable by the processor(s) of the controllermay be configured to perform any of the methods described herein. Forexample, the set of instructions may be configured to apply the pulsedelectrical treatment to de-nucleate cells within the tissue (e.g., skintissue). Thus, the set of instructions may control the timing(frequency, rate, duty cycle, etc.) of the applied electricalstimulation and/or the contact with the tissue.

For example, described herein are methods of treating a skin disordergenerally comprising: applying a treatment to a region of the skin tomake epidermal cells within the region non-viable (e.g., by destroyingor degrading their nuclei and/or other internal organelles, typicallywithout disrupting their cell membranes), without provoking asubstantial inflammatory response, so that the skin forms a necroticcrust over the treated region of the skin and forms new epidermis belowthe necrotic crust so that when the necrotic crust is removed the newepidermis is exposed, while sparing the adjacent dermal tissue.Described herein are also systems configured to apply a treatment to aregion of the skin to make epidermal cells within the region non-viable(e.g., by destroying or degrading their nuclei and/or other internalorganelles, typically without disrupting their cell membranes), withoutprovoking a substantial inflammatory response, such that the skin formsa necrotic crust over the treated region of the skin and forms newepidermis below the necrotic crust so that when the necrotic crust isremoved the new epidermis is exposed, while sparing the adjacent dermaltissue. Alternatively, in some variations, the treatments describedherein may be configured to provoke an immune response, particularly innon-dermal applications. All or some (e.g., greater than 60%, greaterthan 70%, greater than 80%, greater than 90%, greater than 95%, etc.) ofthe epidermal cells within the region may be made non-viable. The regiontypically includes a region around and/or between the electrodesapplying the energy. For example, the region may include a regionapproximately 0.5 mm³ around the electrodes, approximately 1 mm³,approximately 1.5 mm³, approximately 2 mm³, approximately 3 mm³,approximately 4 mm³, approximately 5 mm³, approximately 10 mm³, etc. Thesize of the region may depend at least in part on the size and spacingof the electrodes, as well as the power applied.

Applying the treatment may comprise applying nanosecond electricalpulses having a pulse duration of between 0.1 ns and 1000 ns to theepidermal cells (e.g., 10 to 500 ns, 100 to 350 ns, 150 to 300 ns,etc.). As mentioned, applying the treatment may include applying anon-thermal treatment that does not disrupt the cell membrane of theepidermal cells.

The electrodes may be surface electrodes or penetrating electrodes.Either surface or inserted electrodes may be applied with a gel on theskin region, including a non-conductive or low-conductance gel;alternatively, a conductive gel may be used in some variations. Applyingthe treatment may comprise inserting a pair of electrodes into theregion of the skin and applying a plurality of high voltage nanosecondelectrical pulses between the electrodes. Alternatively or additionally,applying the treatment may comprise applying a pair of electrodesagainst the region of the skin and applying a plurality of high voltagenanosecond electrical pulses from the electrodes.

As mentioned above, applying the pulsed electrical treatment maycomprises applying a plurality of pulses each having a duration ofbetween 0.1 ns and 1000 ns and a peak field strength of at least 1 kV/cm(e.g., duration of between 10 to 500 ns, 100 to 350 ns, 150 to 300 ns,etc., and a field strength of between 1 kV/cm and 50 kV/cm, e.g.,between 10 kV/cm and 40 kV/cm, between 20 kV/cm and 30 kV/cm, etc.). Thetreatment may be applied for less than 5 minutes (e.g., less than 1minute, less than 30 seconds, less than 20 seconds, etc.).

Applying the treatment typically does not result in substantialinflammation. In other words, applying the treatment as described hereintypically increases a marker of inflammation within the region of theskin by less than 15% (e.g., less than 14%, less than 13%, less than12%, less than 11%, less than 10%, less than 9%, less than 8%, less than7%, less than 5%, etc.), wherein the marker of inflammation is one or ofmore of: fibroblast density, leukocyte density, Interleukin-6,Interleukin-8, Interleukin-18, Tumor necrosis factor-alpha, andC-reactive protein.

The skin disorder may be one or more of: seborrheic keratosis, keloids,molluscum contagiosum, sebaceous hyperplasia, syringoma, congenitalcapillary malformation (port-wine stain), melasma, actinic keratoses,dermatosis papulosa nigra, angiofibroma, and warts.

For example, a method of treating a patient's skin may include:positioning a pair of electrodes in communication with a region of apatient's skin; non-thermally destroying the viability of epidermalcells within the region of the patient's skin by applying a plurality ofhigh-field strength, ultra-short electrical pulses to the region of thepatient's skin from the pair of electrodes, without provoking asubstantial inflammatory response within the region, so that the skinforms a necrotic crust over the region of the skin and forms newepidermis below the necrotic crust so that when the necrotic crust isremoved the new epidermis is exposed; wherein the high-field strength,ultra-short electrical pulses comprise a plurality of pulses each havinga duration of between 0.1 ns and 1000 ns and a peak field strength of atleast 1 kV/cm. As mentioned, non-thermally destroying the viability ofepidermal cells within the region of the patient's skin may comprisedisrupting the viability of greater than 90% of the epidermal cellswithin the region of the patient's skin, wherein the region comprises atleast a 1 mm³ volume around the portion of the patient's skin in contactwith the electrodes.

Positioning the pair of electrodes may comprise positioning the pair ofelectrodes in communication with a region of skin having one or more of:a seborrheic keratosis, a keloid, a molluscum contagiosum, a sebaceoushyperplasia, a syringoma, a congenital capillary malformation (port-winestain), a melasma, an actinic keratosis, a dermatosis papulosa nigra, anangiofibroma, a wart, and a tattoo. Other and further features andadvantages of the present disclosure will become apparent from thefollowing detailed description when read in view of the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the apparatuses and methods described herein areset forth with particularity in the claims that follow. A betterunderstanding of the features and advantages of these apparatuses andmethods will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 is an example of a system for generation and deliveringelectrical nano-pulses to a skin lesion.

FIG. 2 is an example of a simplified diode pulse generator.

FIG. 3 is an example of an electrical pulse generated by the system,such as that shown in FIG. 1.

FIG. 4 is an example of an applicator tip with one delivery electrodeand four ground electrodes.

FIG. 5 is an example of a protocol illustrating the use the methodsdescribed herein to treat the skin of a patient. This study was used toillustrate dose ranging over a 60 day time course.

FIG. 6A is an example of untreated (control) skin showing epidermalcells stained to show nuclei.

FIG. 6B is a histological example of rapid de-nucleation with minimalinflammation in the treated tissue. In FIG. 6B, the treated epidermalcells appear as “ghost cells” in which the nuclei are missing; thisimage is taken one day post treatment. The tissue is human skin(abdominal skin) tissue. FIG. 6C shows treated skin seven days aftertreatment; the original epidermis, including the de-nucleated cells, hasformed a necrotic crust that is peeling off of the newly formedepidermis.

FIGS. 7A-7B illustrate the rapid recovery of melanocytes in the newlyformed epidermis in treated skin. In FIG. 7A, a histological section oftreated human skin is shown fifteen (15) days after treatment, showingan initial lack of melanocytes. Melanocytes rapidly recover within 60days post treatment, as shown in FIG. 7B (showing 9 distinctmelanocytes, similar to nearby untreated/control skin). The rapidrecovery of melanocytes is highly predictive of normal melaninproduction and full recovery of skin tone. The lack of a robustinflammatory response following treatment may enhance melanocyterecovery. Typically, injuries with a high amount of inflammation createa higher risk of long-term melanocyte suppression.

FIGS. 8A and 8B show the restoration of elastin distribution andorientation following treatment. As shown in FIG. 8A, initially (e.g.,15 days post-treatment) there is some change in elastin near the surfaceof the skin, however, as shown in FIG. 8B, by 60 days post-treatment theelastin orientation and density is normal compared to nearby (e.g.,control) tissue.

FIGS. 9A-9E illustrate a time course of human skin treated as described,de-nucleating epidermal cells within the region shown, without provokinga substantial inflammatory response. FIG. 9A shows skin one dayfollowing treatment. In FIG. 9B, the same region of skin is shown sevendays post-treatment, showing a necrotic crust formed over the region ofthe treated skin. By 15 days post-treatment (FIG. 9C) the outwardappearance of the skin is improving. FIG. 9D shows the same region ofskin 30 days post treatment. By 60 days post-treatment (FIG. 9E), theskin has recovered and the exposed new epidermis appears nearlyidentical to the nearby untreated normal skin.

FIG. 10A shows a section through a control (untreated) region of theskin. FIG. 10B shows a section through the treated region of the skinone day after treatment, showing the de-nucleated epidermal cells withinthe region and no significant inflammation (e.g., no leukocytes or aleukocyte density comparable to that of control/untreated skin). FIG.10C shows a section through treated skin seven days following treatment,showing the necrotic crust over the newly formed epidermal cells havinghealthy nuclei.

FIGS. 11A-11D illustrate a method of treating skin to remove a lesion,shown as a mole in FIG. 11A. FIG. 11B show a section through a region ofskin including a lesion. FIGS. 11C and 11D prophetically illustrate theremoval of the lesion by treating the skin as described herein. In FIG.11C the lesion is shown as part of the treated skin, in which theepithelial cells are regionally and specifically de-nucleated withoutinvoking a substantial immune response and/or swelling. In FIG. 11D thelesion is separating from the skin as part of the necrotic crust,revealing newly formed epithelial cells beneath the necrotic crust thatdoes not include the lesion.

FIGS. 12A-12H illustrate a method for treating skin (using a pulsedelectrical treatment such as nano-pulse stimulation) to treat a regionof the skin having a seborrheic keratosis. The skin in this example istreated using an applicator such as the one shown in FIG. 121 having aplurality of needle-like electrodes extending from a base region so thatthe ultra-short, high-field strength electric pulses may be deliveredbetween the electrodes. FIG. 12A shows the region of the human skinincluding the seborrheic keratosis prior to treatment. FIG. 12B showsthe lesion immediately following delivery of the nano-pulse stimulation(in this example, 100 pulses of ultra-short, e.g., 100 ns, high-fieldstrength, e.g., 30 kV/cm, electric pulses were delivered over 50seconds) in order to de-nucleate the epithelial cells in the region.FIG. 12C shows the same region of skin one hour after treatment. By 18hours post treatment the necrotic crust has begun forming, which is alsovisible in FIG. 12E. One week following treatment (FIG. 12F), thenecrotic lesion has fallen off (or otherwise been removed), exposing thenew skin forming. FIGS. 12F and 12G show the resulting skin after twoweeks and three weeks, respectively.

FIGS. 13A and 13B illustrate the skin appearance after healing in athermally (e.g., cryosurgically) treated skin region. FIG. 13A shows theuntreated skin, while FIG. 13B shows the treated skin following 90 dayspost treatment, showing discoloration and visible marking.

FIGS. 14A and 14B illustrate skin treated with a pulsed electricaltreatment to de-nucleate epidermal cells within the region withoutprovoking a substantial inflammatory response (e.g., using a nano-pulsestimulation, e.g., ultra-short, high-field strength electric pulses).FIG. 14A shows the skin 15 days after treatment. In contrast, FIG. 14Bshows the same region of skin 60 days post-treatment after removal. Incontrast to the thermal method shown in FIGS. 13A and 13B, thenon-thermal treatment shown in FIGS. 14A and 14B result in significantlymore normal-looking skin, having less discoloration and scarring.

FIGS. 15A-15D illustrate an applicator hand piece (FIG. 15A) andexemplary electrode tips (FIGS. 15B-15D) for an apparatus for treatingskin by delivering nano-pulse stimulation as described herein. The tipsshown in FIGS. 15B-15D may be attached to the end of the applicator ofFIG. 15A. FIGS. 15B and 15C show needle electrodes, while FIG. 15D showsan example of a non-penetrating (plate) electrode. The hand piece shownin FIG. 15A may plug into a generator.

FIG. 16A is a graph illustrating the average epidermal cellnon-viability across all energy treatment levels applied. Error barsindicate the standard error of the mean. The graph illustrates availabledata from eight patients with all treatment levels of the varyingsettings and tips as described herein.

FIG. 16B shows the average adnexal (e.g., dermis) structure effects ofdifferent treatment levels applied. Error bars indicate the standarderror of the mean. In FIG. 16B, the graph illustrates available datafrom eight patients with all treatment levels of the varying settingsand tips.

FIG. 16C illustrates the lack of effect on the average elastin integrityof all of the treatment levels examined. Error bars indicate thestandard error of the mean.

FIG. 16D illustrates average melanocyte count following nanosecondelectrical puling as described herein across different treatment levels.Error bars indicate the standard error of the mean.

FIG. 16E is a graph showing the average dermal inflammation score atvarious post-treatment times, across all treatment levels examined.Error bars indicate the standard error of the mean.

FIG. 17 is a graph illustrating an example of the efficacy of rotatingthe electrodes during treatment of skin, showing that rotating theelectrodes during treatment resulted in similarly sized lesions (inwidth and length) while requiring substantially fewer pulses.

FIG. 18 is a graph illustrating the efficacy of rotating the electrodesduring treatment of non-skin tissue, such as kidney tissue (e.g., pigkidney), also showing that rotating the electrodes at some point duringtreatment resulted in similarly sized lesions (in width and length)while requiring substantially fewer pulses.

FIG. 19 is a graph illustrating the efficacy of rotating the electrodesduring treatment of non-skin tissue, such as liver tissue (e.g., pigliver), also showing that rotating the electrodes at some point duringtreatment resulted in similarly sized lesions (in width and length)while requiring substantially fewer pulses.

FIGS. 20A-20F schematically illustrate a first example of an electricalapplicator (e.g., hand piece with tip) for delivering energy asdescribed herein, including (but not limited to) the delivery ofnano-pulse stimulation. In FIG. 20A the applicator including the tip isshown with the electrodes (needle electrodes) arranged in a firstconfiguration; FIG. 20B shows a front view of the device, showing thefirst position of the electrodes. The electrodes may be rotated by apredetermined amount, as shown in FIGS. 20C and 20D, showing side andfront views, respectively. FIGS. 20E and 20F show side and front views,respectively, of the electrical applicator of FIGS. 20A-20D after thetip has been rotated 90 degrees.

FIG. 21A is another example of an applicator hand piece and exemplaryelectrode tips (FIGS. 21B-21C) for an apparatus for treating tissue bydelivering nano-pulse stimulation and that rotates the electrodesrelative to the tissue by a predetermined amount partway through thetreatment, as described herein. The tips shown in FIGS. 21B-21C may beattached to the end of the applicator of FIG. 21A.

FIGS. 22A and 22B illustrate an example of an applicator having an arrayof needle electrodes that is configured to be activated to achieve thebenefits of the rotation without actually requiring movement of theelectrodes. As shown by example, a pattern of active electrodes ischanged partway through a treatment from the pattern shown in FIG. 22Ato the pattern shown in FIG. 22B, for example.

FIGS. 23A and 23B illustrate another example of an applicator having anarray of needle electrodes that is configured to be activated to achievethe benefits of the rotation without actually moving the electrodes.

FIGS. 24A and 24B illustrate another example of an applicator having anarray of needle electrodes that is configured to rotate the pattern ofthe electrodes on the tissue without having to move the electrodes byactivating different subsets of the electrodes.

FIGS. 25A, 25B, 25C and 25D illustrate front views of a tip of anapplicator that may rotate a pattern of electrodes by electrically ormechanically switching/activating different sub-sets of the electrodesto achieve an effect of rotation of the pattern relative to a tissueregion by +/−45 degrees or 90 degrees, similar to FIGS. 22A-24B. Thepattern of active electrodes shown in FIG. 25A is rotated by −45 degreesin FIG. 25B and by 90 degrees in FIG. 25C, and by 45 degrees in FIG.25D.

FIG. 26 illustrates an example of a robotic or semi-robotic system thatmay be used for rotating a pattern of electrodes partway through atreatment. The robotic system may accurately and precisely controlrotation of the applicator tip during treatment, which may includeinserting the electrode (e.g., needle electrode) pattern into thetissue, applying some portion (e.g., 20%, 30%, 40%, 45%, 50%, 55%, 60%,70%, etc.) of the treatment, then withdrawal from the tissue, andre-insertion into the tissue to apply the remaining portion of thetreatment.

FIG. 27 is a flow chart illustrating one example of a general method ofrotating a pattern of electrodes relative to a tissue region.

FIG. 28 is a flow chart illustrating one example of a method of rotatinga pattern of electrodes relative to a tissue region, for example,without removing the electrodes from the tissue.

DETAILED DESCRIPTION

In general, described herein are methods and apparatuses for treatingtissue (including, but not limited to skin and skin lesion, e.g., toremove or reduce the skin lesion) by applying pulsed electric treatmentto a region of the tissue to make cells within the region non-viable(e.g., by destroying or degrading their nuclei and/or other internalorganelles, typically without disrupting their cell membranes), withoutprovoking a substantial inflammatory response.

The methods and apparatuses described herein may be used to treat tissueby generally applying a treatment, such as pulsed electrical treatment,to the tissue. In some implementations, these methods include performingthe treatment (e.g., treatment dose) in two or more portions deliveredby a plurality of electrodes arranged in a pattern and rotating thepattern of electrodes used to deliver the treatment between thedifferent portions. Although the treatment may be performed or dividedup in this manner, the treatment is still considered a single treatment(or treatment dose), as the second part of the treatment is typicallyapplied immediately or nearly immediately (e.g., within 1 second, 10seconds, 15 seconds, 20 seconds, 30 seconds, 1 minute, etc.) of theapplication of the first part of the treatment. In addition, the energyapplied during the second portion is applied to the same portion of thetissue as the energy applied during the first portion.

The use of apparatuses and method in which the electrodes are rotatedbetween different parts of the treatment are described in greater detailbelow, in reference to FIGS. 17-26.

Although in general, the methods and apparatuses described herein may beused to treat any tissue, many of the examples and illustrationsdescribed herein include skin tissue for convenience of description.

Skin Treatment

When applied to skin, epidermal cells may be destroyed by destroyingtheir nucleus so that the skin forms a necrotic crust over the region ofthe skin and forms new epidermis below the necrotic crust so that whenthe necrotic crust is removed the new epidermis is exposed. Although themethods described herein are described in the context of non-thermalde-nucleating epidermal cells within the region, other methods of makingthe epidermal cells within the region non-viable may be used, includingdisrupting or destroying other organelles in the epidermal cells, suchas the endoplasmic reticulum, mitochondria, etc. The non-thermaltreatment may be nano-pulse stimulation (e.g., ultra-short, high-fieldstrength electric pulses) adapted to de-nucleate epidermal cells withoutprovoking an inflammatory response (e.g., without increasing the densityof leukocytes and/or melanocytes above a threshold percentage comparedto untreated skin).

Illustrative embodiments are now discussed. Other embodiments may beused in addition or instead. Details which may be apparent orunnecessary may be omitted to save space or for a more effectivepresentation. Conversely, some embodiments may be practiced without allof the details which are disclosed.

In any of the methods described herein, the pulsed electrical treatmentmay be nano-pulse stimulation, which may include the application ofelectrical pulses with duration of 1,000 nanoseconds (ns) or less asmeasured, for example, at the full-width-at-half-maximum (FWHM) of thepulse wave.

A skin lesion that may be treated by the devices described herein may beany deviation of skin from a healthy or a normal condition. Examples ofthe skin lesions include skin diseases, conditions, injuries, defects,abnormalities or combinations of thereof. For example, such skin lesionsmay be malignancies (such as basal cell carcinomas, squamous cellcarcinoma and melanoma), precancerous lesions (such as actinickeratosis), human papilloma virus (HPV) infected cells (such as verrucavulgaris or common warts, plantar warts, genital warts), immune-relatedconditions (such as psoriasis), other skin abnormalities (such asseborrheic keratosis and acrocordon) and combinations thereof. The skinlesion may also include aged skin, wrinkled skin or damaged skin. Anexample of the damaged skin is the skin damaged by sun radiation. In oneembodiment, the skin lesions may be basal cell carcinoma (includingpapilloma), squamous cell carcinoma, actinic keratosis, warts, orcombinations thereof. In one embodiment, the skin lesion may be a skinlesion of a human. In this embodiment, the skin lesion may comprisebasal cell carcinoma, squamous cell carcinoma, actinic keratosis, warts,or combinations thereof. In this embodiment, the skin lesion may alsocomprise common warts, actinic keratosis, or combinations thereof. Theskin lesion may be a common wart of a human. The skin lesion may also bean actinic keratosis of a human.

The pulsed electrical treatment, such as a nano-pulse stimulationtreatment, may be achieved by providing electrical energy to the skinlesion in a form of one or more electrical pulses. During thistreatment, tissue removal may not be intentional and, if it happens, maynot be substantial. Thus, the treatment may thereby be advantageous overcurrent or other proposed treatment techniques since it may achieve itspurpose with no substantial tissue removal. Further, these methods maybe generally non-thermal, and may be configured to prevent substantialinflammatory response.

The treatment of the skin lesion may prevent growth of the lesion. Thetreatment may reduce the volume of the skin lesion. That is, thetreatment induces at least shrinkage of the lesion. This shrinkage maybe at least 10%, 20%, 30%, 60%, 70%, 80%, 90%, or more than 90%. Thetreatment may reduce the skin lesion volume to a negligible level (i.e.,clearance of the lesion). The lesion growth prevention or the lesionvolume reduction may be achieved in at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or more than 90% of cases.

When the lesion volume shrinks to a negligible size (i.e. about 100%),the lesion is “cleared”. If the lesion growth or shrinkage is less than10% after the treatment, the lesion growth is considered to have been“prevented” or that there is “no change”. If the lesion shrinkage is inthe range of >10% and <50%, it is concluded that there is lesion“shrinkage”. If the lesion shrinkage is in the range of >50% and <100%,it is concluded that there is “substantial shrinkage”. If the lesiongrowth is in the range of >10% to <100%, it is concluded that there islesion “growth”. And if the lesion growth is >100%, it is concluded thatthere is “substantial growth”.

If the height (i.e. protrusion) of the lesion above the skin surface isnegligibly small, i.e. about 0.00 mm, the lesion height is recorded asabout 0.10 mm.

The treatment results may be permanent or temporary. In one embodiment,the growth prevention, or the shrinkage or the clearance may last for aduration of at least 7 days, at least 10 days, at least 20 days, atleast 30 days, at least 40 days, at least 50 days, at least 60 days, atleast 70 days, at least 80 days, at least 90 days, at least 100 days, orat least 110 days.

The treatment may comprise at least one treatment session. For example,the treatment session may comprise an administration of the electricalenergy to the skin lesion of a human by physician at an office visit.The treatment of a human lesion may also comprise a plurality oftreatments sessions. For example, it may comprise at least two treatmentsessions or at least three treatment sessions. These treatments may becombined with any other treatment to increase efficacy of the lesiontreatment. These other treatments may include over-the-countertreatments, treatments with prescription medicines, surgery, anddestructive procedures. For example, these other lesion treatments mayinclude curettage, electrodessication, cryotherapy, topical therapy, andcombinations thereof.

When the pulsed electrical treatment is nano-pulse stimulation, anysystem suitable for delivery of electrical nano-pulses with a durationof 1,000 ns or less to the skin lesion may be used. The system maycomprise a power supply, a controller, a pulse generator, and a pulsedelivery device (e.g., a wand). An example of this system isschematically shown in FIG. 1.

The pulse generator 101 may be any pulse generator that is capable ofgenerating pulses with a duration of 1,000 ns or less at FWHM. The pulsedelivery device may be any device that can deliver electrical pulses tothe skin lesion. This device may have an applicator tip that maycomprise at least one delivery electrode. This applicator may furthercomprise at least one ground electrode. The delivery electrode and/orthe ground electrode may penetrate into the skin lesion to deliver theelectrical pulses. The delivery electrode and/or the ground electrodemay deliver the electrical pulses without substantially or intentionallypenetrating into the skin lesion. For example, the skin lesion may beconstricted between the electrodes or the electrodes may only touch thelesion (or the region surrounding the lesion) during the delivery of theelectrical pulses.

A system for treating tissue (e.g., skin) as described herein mayinclude an applicator 103 having a treatment tip 105 with two or more(e.g., a plurality) of electrodes. The system may generally include acontroller 107. The controller may control operation of the system, andmay include one or more processors, one or more memories, and the like.The controller may include logic (e.g., hardware, software, firmware)including instructions that, when executed by the one or moreprocessor(s), may control the system to apply the electrical therapy asdescribed herein. For example, the set of instructions may operate arobotic actuator (e.g., robotic arm) to move the treatment electrodes tothe target tissue region and control the application of pulsedelectrical energy treatment to the tissue. The set of instructions mayinclude instructions controlling the application of the pulses, rotationof the pattern of electrodes applying the energy and/or placement of theapplicator on/off of the tissue.

For example, in some variations, the system may include a means forcontrolling the applicator and a means for applying the pulsedelectrical energy. The system may also include a means for applyingenergy to denucleate cells within the target treatment tissue (e.g.,skin). Any of these systems may also include a means for rotating thepattern of electrodes partway through the treatment.

An example of an applicator tip is illustrated in FIG. 4. In thisexample, the applicator tip has one delivery electrode placed at thecenter and four ground electrodes surrounding the delivery electrode.The base of the electrodes may be embedded in a solid insulatingmaterial to maintain separations between them.

The electrical energy may be applied to the skin lesion in the form ofat least one electrical pulse. For example, between 30 and 150 pulsesmay be applied (e.g., between 33 and 100). In one embodiment, at least10 pulses, at least 100 pulses or at least 2000 pulses (e.g., at least1,000) pulses may be applied to treat the lesion during a singletreatment. The duration of one or more of the pulses may be in the rangeof 0.01 ns to 1,000 ns. For example, the pulse width may be between 100and 500 ns (e.g., between 200 and 300 ns). The duration of one or moreof the pulses may be, for example, in sub-microsecond range, or in therange of 1 ns to 600 ns, or in the range of 1 ns to 300 ns.

The total electrical energy applied per volume of skin lesion may be atleast 10 mJ/mm³, at least 20 mJ/mm³, at least 100 mJ/mm³, at least 500mJ/mm³, or at least 1,000 mJ/mm³. In another embodiment, the totalapplied electrical energy per volume of the skin lesion may be in therange of 10 mJ/mm³ to 10,000 mJ/mm³.

The electrical field produced by each pulse may be at least 1 kV/cm atthe peak amplitude of the pulse. For example, the electrical field maybe between 10 and 50 kV/cm (e.g., between 20 to 30 kV/cm). Theelectrical field produced by each pulse may also be at least 10 kV/cm atthe peak amplitude of the pulse. In another embodiment, the electricalfield produced by each pulse may be in the range of 1 kV/cm to 1,000kV/cm at the peak amplitude of the pulse. Yet, in another embodiment,the electrical field produced by each pulse may be in the range of 10kV/cm to 100 kV/cm at the peak amplitude of the pulse.

The treatment may comprise at least one treatment session, i.e.administration of the electrical energy to the skin lesion by physicianat an office visit. This treatment session may comprise at least oneapplication of the electric energy to a lesion. The electrical energymay be delivered to the skin lesion in any manner suitable for the skinlesion. For example, the electrical energy may be delivered aftercontacting the surface of the lesion by electrodes of the applicatortip. In this example, the electrodes don't penetrate into the lesionduring the application of the electrical energy. In another example, theelectric energy may also be delivered after insertion of the electrodesto the skin lesion. For example, one application may comprise firstpenetration of the skin lesion by the electrodes of the applicator tipand then delivery of a desirable number of pulses, for example, about100 pulses, with a pulse duration of between about 200 to 300 ns. Morethan one application may be used per treatment session to treat thelesion. The number of applications may depend on the size and/or thetype of the lesion. Larger lesions may require more than one applicationper treatment session, as discussed in detail below. Also, differenttypes of lesions may require higher energies, and therefore moreapplications per treatment session may be needed to at least prevent thegrowth of the lesions. The treatment of a lesion may also comprise aplurality of treatment sessions. For example, it may comprise at leasttwo treatment sessions or at least three treatment sessions. Thesetreatment sessions may also be separated in time by 1 or more days(e.g., 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.).

An electrical pulse generation and delivery system is schematicallyshown in FIG. 1 and includes a pulse generator 101. An example of thepulse generator is schematically shown in FIG. 2. As shown in FIG. 2,the diode pulse generator may include a tank circuit consisting ofinductances L1 and L2 and capacitances C1 and C2. The tank circuit maybe connected in series with a diode D across which a load RL to bedriven may be connected. This load may be the resistance of the lesionor tissue. The pulse generator may include a switching system, such asswitches S1 and S2, which may be electronic. A voltage supply V_(in) maybe connected to the diode pulse generator through a resistance R_(ch).Other examples of pulse generators and systems that may be used with anyof the methods of the present disclosure and/or may be modified to formany of the apparatuses described herein are shown and described inco-pending U.S. patent application Ser. No. 15/148,344, U.S. patentapplication Ser. No. 15/269,273, U.S. patent application Ser. No.15/595,684, U.S. patent application Ser. No. 15/347,729, U.S. patentapplication Ser. No. 15/444,738, and U.S. patent application Ser. No.15/347,728; each of these patent application is herein incorporated byreference in its entirety.

Before the beginning of a pulse cycle, the switch S1 may be open and theswitch S2 may be closed. This may cause the capacitance C1 to fullycharge and the capacitance C2 to fully discharge. At the beginning ofthe pulse cycle, the switch S1 may be closed and the switch S2 may beopened. This may cause charge to transfer from the capacitance C1 to thecapacitance C2. During this transfer, the current through the tankcircuit may rise and fall in approximately a sinusoidal manner.

This current may cause the diode D to be forward-biased as it travelsthrough it. During this process, charge may be stored in the depletionlayer of the diode D. At the end of the half-cycle, switch S2 may beclosed. During the next half-cycle, the current flow may reverse indirection, causing the diode D to be reverse-biased. During the firstpart of the second half-cycle, current may still flow through the diodeD while charge in its depletion layer is being depleted. Once the chargeis depleted, the current through the diode D stops, causing the diode toappear as an open switch. This may cause the current through theinductance L2 to commute from the diode D to the load RL. The diode Dmay thus be configured to act as an opening switch, interrupting thecurrent in the inductance L2 and commuting it into the load RL. Currentmay now travel through the load RL until the energy stored in the tankcircuit consisting of the capacitance C2 and the inductance L2 depletes,thus delivering a pulse into the load RL.

This pulse generator included a current limiting resistor, RCLconfigured to limit damage to the pulse generator. The value of thisresistor was about 1 ohm. The pulse generator further included aterminating resistance, RT in parallel with the diode, wherein theterminating resistance was configured to protect the output stage of thepulse generator. The value of this resistor was about 100 ohms. Thepulse generator disclosed above may provide at least one electricalpulse with a duration varying in the range of about 7 nanoseconds (ns)at FWHM to about 20 ns at FWHM. In one example, a pulse with duration ofabout 20 ns at FWHM was generated. The characteristics of this pulsewere recorded. As shown in FIG. 3, this pulse had pulse duration ofabout 20 ns at FWHM and a peak amplitude of about 8.00 kV.

The electrical nano-pulses were delivered to a lesion by usingapplicator tips comprising one delivery electrode and four groundelectrodes surrounding the delivery electrode. This applicator tip isshown in FIG. 4. Each electrode was constructed by using a 30 gaugeneedle (i.e. about 0.255 mm in diameter). The delivery and the groundelectrodes have the same length for each applicator tip. This lengthvaried in the range of about 2 millimeters (mm) to 5 mm. The electrodeswere placed to form a square pattern. The ground electrodes were at thecorners of this square and the delivery electrode was at its center.Center-to-center distance between the delivery electrode and each groundelectrode was about 1.75 mm. This configuration provided a volume ofabout 30.625 cubic-millimeters (mm3) within the boundary formed by theground electrodes. The ground electrodes and the delivery electrode wereelectrically isolated from each other by embedding them in a Tefloninsulation (not shown in FIG. 4).

The tip configuration may be different than illustrated. There may beother applicator tip configurations suitable for the treatment of thelesions. These configurations may include tips comprising at least onedelivery electrode and at least one ground electrode. For example, asthe system disclosed above is coaxial in nature, with the groundelectrodes surrounding the delivery electrode, any number of needleconfigurations may be realized, including a circular arrangement withfive or more ground electrodes, a triangular arrangement with threeground electrodes, wherein the delivery electrode may be placed at thegeometrical center of such arrangements. A simple linear arrangementwith just two opposing electrodes, i.e., one return electrode and onedelivery electrode, may also be used for the delivery of the electricalpulses.

Still other tip configurations, for example those with differentelectrode spacing or length, may also be used for the treatment of thelesions. However, as the effect of these short pulses on cells islargely dependent upon the strength of electric field, an increase inreturn and active electrode spacing may have to be accompanied by aproportional increase in output voltage to maintain the required fieldfor the effect on cells. Similarly, if the spacing is reduced, thevoltage could be proportionally decreased.

Each pulse with a duration of about 7 ns at FWHM in this example mayinclude a carrier frequency. For example, a pulse may containsignificant frequency components centered at about 142.9 megahertz(MHz), and each pulse with a duration of about 14 ns at FHWM containedsignificant frequency components centered at about 71.4 MHz. Electricalnano-pulses with different amplitudes (e.g., peak amplitude of about 7.0kilovolts (kV), peak amplitude of about 5.5 kV, etc.) may be used. Inone example, the electrical field is about 40 kilovolts/centimeter(kV/cm) at the peak amplitude of about 7.0 kV and about 31 kV/cm at thepeak amplitude of about 5.5 kV.

Values of the pulse durations and the peak amplitudes referred to hereinmay be average values unless specifically noted. These pulse durationsand the peak amplitudes may vary with a standard deviation of 10% oftheir average values. For example, the pulse duration of about 7 ns atFWHM may be an average of pulse durations that vary within the range of6.30 ns and 7.70 ns, or it is 7.00±0.70 ns. Similarly, the peakamplitude of about 7.00 kV may be an average of the peak amplitudes thatvary within the range of 6.30 kV and 7.70 KV, or it is 7.00±0.70 kV. Thelesion resistance may be expected to be about 100 ohms. In general, theskin impedance values may be related to the design of the electrodebeing used. For example, see the electrodes shown in FIGS. 15A-15D anddescribed in detail below. Different electrode designs may registerdifferent tissue impedances, e.g., between about 100 Ohms and 1 KOhm(e.g., from 150 Ohms to 800 Ohms), depending on the quality of theelectrode contact, which may be (in part) a function of the electrodedesign.

An abdominoplasty study was done on human subjects to evaluate sixtreatment levels (TLs, low to high) on human skin using differenttreatment applicators (treatment tips), including 5 mm×5 mm and 2.5mm×2.5 mm tips (similar to those shown in FIGS. 15B and 15C). Abdominalskin was treated in five patients at 1, 5, 25, 30 and 60 days before thetissue was removed, and histological examination of the treated (andnearby control) sites was performed. FIG. 5 illustrates the pattern ofstimulation applied to the abdominal skin during the trial on eachpatient. Treatment date 1 was performed 60 days before removal andanalysis of the skin tissue. For each treatment date a 3×2 grid oftreated tissue was treated, and each treatment site was treated with adifferent treatment level (TL1, TL2, TL3, TL4, TL5 and TL6, inincreasing levels). This protocol provided a time course of healing ofeach of the six treatment levels from 1 to 60 days after exposure. 8 mmdiameter discs of tissue including each treatment site was then biopsiedand examined. These treatment levels were ranked from decreasing toincreasing intensity levels. The treatment levels each had an electricfield strength between 20 to 30 kV/cm, a pulse width between 200 to 300ns, a frequency between 2 to 6 Hz, and between 33 to 100 pulses.Generally, the energy per pulse, as well as the total energy applied bythe electrodes, for each treatment level increased from TL1 to TL6.

Based on the preliminary results from this work, treatment levels inwhich the skin was stimulated sufficiently so that dermal cells withinthe epidermal region being treated were de-nucleated and for whichlittle or no inflammatory response was seen resulted in the formation ofa necrotic crust over a region of new epidermis which, by 60 days,formed new skin with very little, if any, discoloration and/or scarring.

As part of the analysis of this study, various tissue stainingtechniques intended to identify specific cellular changes and tissuemorphology were utilized in the histologic analysis to characterize theinitial tissue responses and the subsequent recovery processes, andsequential findings for each of the six energy levels were compared tonormal control punch biopsies in the same patient. This study found thatthe method of using low energy nano-pulse cellular stimulation on skinas described herein lead to a loss of viability of the epidermal layerof skin (at all energy levels examined for treatment) by at least thefirst day post-treatment. There was a lack of observed effect on dermalcollagen, and little, if any inflammation given the amount of epidermaldamage. This lack of inflammatory effect is consistent with preservationof fibroblasts, elastin, and melanocyte recovery. The transient effecton deeper cellular structures in the dermis suggest a surprisingaffinity for highly cellular tissue, and a sparing effect on the lesscellular connective tissue of the collagen layer.

FIG. 16A summarizes the effect of the stimulation across all of theenergy levels tested above on epidermal cells. A review of the changesof the entire thickness of the epidermal/dermal layer and subcutaneousfat from day 1 through day 60 was performed. The primary changes due tothe non-thermal energy exposure were observed in the treated skin withinthe epidermal layer of skin. At day one post-treatment, tissue samplesfrom all subjects showed evidence of ghost cells in the epidermal layercharacterized by intact cell membranes and absence of nuclei withinthose cells, which indicates a non-viability of those cells. Thenon-viability of epidermal cells was full-thickness and complete for allenergy settings by 1 day post-treatment. In many patients, hairfollicles and eccrine glands within the dermal layer of the skin werealso visible for histologic review. In specimens in which hair folliclesand eccrine ducts were visible, there was a partial (50%) to fullnecrosis of the upper portions of the hair follicles and eccrine ducts.Five (5) days post-treatment epidermal changes range from 50% to 90%healing (50%-10% non-viable epidermal cells), with the exception of 3patients of the highest treatment energy. Hair follicles and eccrineducts when visible in the tissue samples showed focal dyskeratotic cellswithin the hair follicles at 5 and 15 days consistent with partialhealing at 5 days and complete recovery by 15 days. Eccrine ducts oftenshowed focal squamous metaplasia, a sign of re-epithelialization. By 15days post-treatment, the epidermis layer had returned to normal inalmost all cases. By 60 days, the epidermis, hair follicles, and eccrineglands had all completely returned to normal. In two patients treated atthe highest energy level, epidermal necrosis was followed by a formationof an inflammatory eschar. This formation healed by 60 days with someepidermal flattening and minimal papillary dermal fibrosis.

FIG. 16A shows a quantitative analysis performed on the epidermal layerof the skin based on a scale rating of the epidermal integrity.Different energy treatment levels were applied to the targeted normalabdominal tissue among the 8 patients treated. FIG. 16A shows thenon-viability of the cells across all different treatment energy levelsover the time period of 1 day to 60 days post treatment (postapplication of nano-pulse stimulation. A score of 100% indicatescompletely non-viable epidermis, a score of 10% indicates recoveredepidermis with evidence of “flatness”, and a score, and score of 0%indicates completely viable epidermal cells (complete epidermalrecovery). Almost all energy levels showed complete non-viability ofepidermal cells by 1 day post-treatment with an average of 88%completely non-viable cells. By 60 days the epidermis, hair follicles,and eccrine glands had all completely returned to normal, with theexception of 1 patient at the highest treatment level.

Minimal alterations in the dermal collagen were observed, with noevidence of thermal injury apparent. In several tissue samples exposedto the highest treatment levels, there was focal papillary dermalnecrosis evident. This effect was observed at the 1 day and 5 dayintervals, but was not observed at the 15-60 day intervals. In twopatients treated at the highest energy level, there was some parallelfibrosis of the dermal collagen bundles at 60 days.

FIG. 16B shows a quantitative analysis on the adnexal structure effectsbased on a scale rating. A score of 0% indicates there is no effect onadnexal structures, a score of 50% indicates that about half of observedadnexal cells are pink and glassy, and a score of 100% indicatesnecrosis of hair follicle. An average score of 33% and 16% was shownacross all treatment levels applied for day 1 and day 5 respectivelywith no adnexal structure effect seen past day 15 for any energy level.

Elastic tissue remained intact in the vast majority of patients. In thehigher treatment levels there was occasional slight decrease in elasticfiber at days 1, 5 and 15, with return of elastic fibers observed bydays 30 and 60. Taken in summary with the results of the trichromecollagen stains, these findings indicate relative minimal effects on thedermis. This predicts a very low risk of scarring.

A quantitative analysis was performed on the elastic integrity based ona scale rating shown in FIG. 16C. A score of 1 value indicates there isno effect on elastin fibers, a score of 2 indicates a mild effect onelastin fibers and loss of elastic fibers in papillary dermis, and avalue of 3 indicates a moderate effect on elastin fibers and loss offibers in reticular dermis. As seen in FIG. 16C there is no apparenteffect on the elastin integrity at any time following treatment.

The number of melanocytes was observed using a MITF immunostain.Staining for melanocytes showed a marked loss of melanocytes in thetreatment areas at day one, however by day 15 the number of melanocytesreturned to normal density, comparable to the control specimens. Thiswas observed through 60 days. The rapid return of the melanocyticdensity to levels comparable to control should be consistent with arelative normalization of skin pigmentation over time. The number ofmelanocytes was quantified throughout the time periods and depicted inFIG. 16D.

The number of dermal fibroblasts appeared to decrease at day 1 and day 5following treatment. However, by day 15, 30 and 60 the number of dermalfibroblasts was equivalent to pretreatment samples. This suggests atemporary decrease in the number of dermal fibroblasts after treatmentand possibly some loss of dermal fibroblasts secondary to the treatment.However, fibroblasts are recruited from normal surrounding skin andperipheral circulation and the population of fibroblasts returned tolevels similar to the control in the 15-60 day time period. This findingsuggests a recovery of fibroblasts and the associated normal capacity torebuild connective tissue.

Epidermal staining was performed using immunohistochemical stains toactive Caspase 3. This analysis shows no significant expression withinthe epidermis in the 1 and 5 day post-treatment time frames tested.These findings do not provide clear evidence of apoptotic cell deathduring the analyzed time frames. Therefore, the precise mechanism forthe observation of ghost cells and associated cell death by 24 hourspost-treatment was not identifiable by this method.

Most interestingly, the degree of dermal inflammation was minimalcompared to other thermal or physical methods of intentionally damagingsurface epidermal tissue. There was a small amount of inflammation seenat day 1 and day 5, however the amount of inflammation appears to besparse and perivascular. Of note, there were several samples whichshowed focal perivascular inflammation with fibrin deposition suggestiveof low grade vascular damage. This can be seen at day 5 through day 60and appeared to have no clinical correlation to degree of epidermalnecrosis or impact on healing.

A quantitative analysis was performed on the average dermal inflammationscores based on a scale rating shown in FIG. 16E. A score of 3 indicatesevidence of fiberplasia and likely long term scar. A score of 2indicates evidence of fibroplasia and unlikely long-term scar and ascore of 1 indicates no evidence of fibroplasia.

In summary, the methods of using low energy nano-pulse cellularstimulation on skin as described herein led to a loss of the epidermallayer of skin by at least one day post-treatment (likely faster). Thesemethods did not affect dermal collagen and resulted in a relatively lowlevel of inflammation given the amount of epidermal damage. This lack ofinflammatory effect is consistent with preservation of fibroblasts,elastin, and/or melanocyte recovery.

For example, FIGS. 6A-6C illustrate histological sections of skintreated with an intermediate treatment level (TL4), showing rapidepidermal layer destruction by de-nucleation with minimal inflammation(e.g., a rise of than about 15% in acute inflammatory markers in thetreated tissue) and rapid re-epithelization. For example, FIG. 6A showsa histological section through a control region of skin adjacent to thetreated regions; the skin has been stained with a nuclear stain andshows dark nuclei in untreated epithelial cells.

FIG. 6B is a stained section though a region treated (TL4) one day priorto fixation and staining. In FIG. 6B the epithelial cells have losttheir nuclei, becoming “ghost cells” in which the cell membrane appearsintact, but the cells 603 are non-viable as their nucleus has beenselectively destroyed by the treatment. It can be seen from therepresentative image in FIG. 6B that there are no cellular indicators ofinflammation (e.g., leukocyte density is unchanged, compared to controlregions). FIG. 6C shows an example of a tissue region seven days posttreatment. By a few days following treatment at this moderate treatmentlevel (TL4), the de-nucleated cell has formed a necrotic crust 605 andby day 7 post-treatment, have begun to peel away from the underlyingregion 609 in which new epidermal cells 607 have formed, showing healthynuclei and little, if any, inflammation.

Any of the methods and apparatuses described herein may also beconfigured to provide for new epidermis having less scarring anddiscoloration by de-nucleating epidermal cells in a treatment zonewithout invoking an inflammatory response (unlike in thermal treatments)so that melanocytes and extracellular matrix are distributed in a mannerthat resembles or replicates nearby ‘normal’ untreated skin. Forexample, FIGS. 7A and 7B illustrate the recovery of melanocytesfollowing a treatment similar to that shown in FIGS. 6B-6C. In FIG. 7A atreated region of skin is shown 15 days following treatment with a highto moderate (TL5) treatment level. As shown in FIG. 7A, shortly aftertreatment (e.g., by 15 days post treatment) there is initially anabsence of melanocytes. FIG. 7B histologically shows that in the treatedskin sixty (60) days following treatment there is an averagedistribution of melanocytes, similar to control skin. In FIG. 7B, theimage shows nine distinct melanocytes 707, a similar density anddistribution to what is shown by nearby control (untreated, lesion-free)tissue. Rapid recovery of melanocytes is highly predictive of normalmelanin production and full recovery of skin tone; injuries with a highamount of inflammation typically create a higher risk of long-termmelanocyte suppression.

FIGS. 8A and 8B illustrate the effect of treatment as described hereinon the distribution and density of elastin near the surface of the newlyformed epidermal tissue. Typically, restoring the elastin to normalresults in a reduction in scarring risk. In FIG. 8A, following a hightreatment level (TL6), after 15 days from treatment, the treated skinshows some change in elastin near the surface of the tissue. However, bysixty days post-treatment, as shown in FIG. 8B, the elastin has beenrestored to a nearly normal orientation and density.

The overall appearance of the skin when treated to de-nucleate theepithelial cells, e.g., using a moderate level (TL4) of nano-pulsestimulation is illustrated in FIG. 9A-9E, showing representative imagesfor different treatment times. In FIG. 9A, one day following treatment,the epithelial cells have de-nucleated without evoking a substantialimmune response. Seven days after stimulation (FIG. 9B), a substantialnecrotic crust 903 has formed on the tissue from the de-nucleated cells.This crust progressively recedes (e.g., is removed or simply falls off),as shown in FIGS. 9C-9E (at fifteen days, thirty days or sixty days,respectively). For side-by-side comparison with the histologicalsections, FIGS. 10A-10C show the sections of FIGS. 6A-6C.

In general, any of the methods and apparatuses described herein may beused to treat skin having a lesion by de-nucleating the epidermal cellscomprising, surrounding or underlying the lesion. For example, FIGS.11A-11D illustrate one method of treating a seborrheic keratosis (SK).SKs are one of the most common noncancerous skin growths on olderadults, appearing as small, rough bumps. FIG. 11A illustrates an exampleof an SK 1103 on a patient's skin, and FIG. 11B is a histologicalsection through a typical SK. The methods described herein may be usedto treat a lesion such as SK as shown in FIG. 11C, propheticallyillustrating what treatment of a region of skin including a simulated SK1103′ in a region of tissue having epidermal cells has been de-nucleated1105 as discussed above, forming “ghost” epidermal cells. FIG. 11Dillustrates the exemplary section of FIG. 11C after seven days fromtreatment, in which the SK lesion 1103′ forms part of the necrotic crust1107 that will be removed from the tissue to expose new epidermis belowthe necrotic crust. In general, the longer the skin has to recover, themore like ‘normal’ skin it will resemble; the lesion may be partially orcompletely removed.

FIGS. 12A-12H illustrate a time course showing the surface of skintreated to remove an SK lesion 1203. In FIGS. 12A-12H, the skin istreated using an applicator such as the one shown in FIG. 121 having aplurality of needle-like electrodes 1207, 1211 extending from a baseregion 1209 so that the ultra-short, high-field strength electric pulsesmay be delivered between the electrodes. FIG. 12A shows the region ofthe human skin including the seborrheic keratosis 1203 prior totreatment. FIG. 12B shows the lesion immediately following delivery ofthe nano-pulse stimulation (in this example, 100 pulses of ultra-short,e.g., 100 ns, high-field strength, e.g., 30 kV/cm, electric pulses weredelivered over 50 seconds) in order to de-nucleate the epithelial cellsin the region. FIG. 12C shows the same region of skin one hour aftertreatment. By 18 hours post treatment the necrotic crust 1205 has begunforming, which is also visible in FIG. 12E. One week following treatment(FIG. 12F), the necrotic lesion has fallen off (or otherwise beenremoved), exposing the new skin forming. FIGS. 12F and 12G show theresulting skin after two weeks and three weeks, respectively.

The methods described herein may generally provide a superiorappearance, e.g., more natural color and/or less scarring, compared toother, particularly thermal, skin treatment methods. For example, FIGS.13A and 13B illustrate recovery following cryosurgical treatment of askin region including a lesion. FIG. 13A shows a region of skinincluding a skin lesion 1303. Three months (90 days) followingcryosurgical treatment of the lesion, the skin remains discolored 1305,as shown in FIG. 13B. By contrast the methods (including the use ofnano-pulse stimulation as described herein) may provide therapies withless discoloration and scarring, as shown in FIGS. 14A-14B. in FIG. 14A,a (simulated) lesion may be removed by de-nucleating the epidermal cellswithout provoking a substantial inflammatory response; the skin showingthe resulting necrotic crust 1403 is shown for 15 days post-treatment(e.g., following a moderately-high treatment level, TL5). By sixty dayspost-treatment, as shown in FIG. 14B, the lesion has been removed (alongwith the necrotic crust), leaving the skin reasonably clear and free ofdiscoloration.

According to one aspect, an apparatus for treating tissue (e.g., skindisorders, skin abnormalities, skin lesions and tumors) is provided. Theapparatus comprises a pulse generator; a set of electrodes; and acontroller configured to control, at least partially, operation of thepulse generator, the controller comprising a processor having a set ofinstructions, wherein the set of instructions, when executed by theprocessor causes the pulse generator to generate and apply through theset of electrodes a pulsed electrical treatment to a region of tissue tode-nucleate cells within the region without provoking a substantialinflammatory response, so that the tissue forms a necrotic crust andforms new tissue below the necrotic crust so that when the necroticcrust is removed the new tissue is exposed. Any of the apparatusesdescribed herein may include a hand-held applicator having a handpiecethat plugs into an applicator. For example, FIG. 15A shows an example ofa handpiece 1501 that may plug (via cord 1503) to a generator (notshown) for generating the nano-pulse stimulation. One or more differenttips may couple with the handpiece; the tips may include theelectrode(s) for delivering the energy to the skin, as described above.For example, FIGS. 15B-15D illustrate exemplary electrode tips fortreating skin by delivering nano-pulse stimulation as described herein.In FIG. 15A, the tip fits over the distal end of the headpiece 1505, andsnaps or locks in place once electrical contact is made with theprojecting (needle-like) electrodes 1511. For example, the tip may bemechanically secured (e.g., by snap-fit, friction fit, etc.) onto theend of the handpiece. In FIG. 15B, two electrodes are provided, and eachis sufficiently sharp so that it may penetrate the skin. One electrodemay be the cathode and the other electrode the anode. The electrodes maybe pointed and/or sharp, or otherwise configured to penetrate thetissue. The region between the electrodes may be adapted to fit over askin lesion that projects from the skin. FIG. 15C shows a tip 1507 thatincludes two parallel rows of sharp, tissue penetrating electrodes thatmay all simultaneously penetrate the skin in the region including orsurrounding the skin lesion. In this example, the electrodes (orelectrode pairs) may be separately addressed by the apparatus, or theymay be connected together. For example, in FIG. 15C, the left row ofelectrode may be electrically connected (e.g., acting as a cathode) andthe right row of electrodes may be electrically connected (e.g., actingas an anode).

FIG. 15D illustrates an example of a non-penetrative (e.g., surface) tip1509 including electrodes that are configured to deliver nano-pulseelectrical energy as described herein. In FIG. 15D, an outer ring ofelectrode surrounds an inner electrode; these electrodes may act as anelectrode pair for delivering energy (e.g., current) to the skin. Thetips in FIGS. 15B-15D may be swapped.

Rotation of Electrode Pattern

Also described herein are methods and apparatuses/systems in which theelectrodes applying the energy are rotated partway through theapplication of the treatment. These methods and apparatuses may be usedto treat any tissue, including (but not limited to) skin. Surprisingly,when compared to an identical treatment in which the electrodesdelivering the pulsed electrical treatment are not rotated, the amountof energy required to treat the tissue (e.g., to achieve a lesion of apredetermined size) is significantly less. The methods and systemsdisclosed herein may generally provide a more efficient treatment, forexample, requiring less energy to be applied to the tissue (e.g., fewerpulses, lower energy pulses, shorter treatment times, etc.) in order toachieve therapeutically desirable treatments. These methods andapparatuses may allow to increase the treatment volume or size andimprove targeting accuracy. The systems and methods of the presentdisclosure may provide, among other benefits, the improved increasedtreatment volume. For example, when the treatment comprises a pluralityof pulses, such as sub-microsecond electrical pulses, the number ofpulses required to achieve a predetermined lesion size when theelectrodes are rotated partway through the treatment as disclosed hereinmay be substantially less than the number of pulses required if theelectrodes are not rotated. For example, the number of pulses, requiredto form an equivalently-sized lesion by pulsed electrical treatment maybe reduced by between about 40% and 20% when rotation (e.g., rotation by90 degrees) is implemented, compared to treatment without rotation ofthe pattern of electrodes applying the pulsed electrical treatment. Inaddition, the methods and systems disclosed herein may provide not onlyfor the larger volume but also for more consistent and uniform volume.Moreover, using in some implementations, automated, including computercontrolled, systems may provide precise and accurate rotating andrepositioning of the energy delivery device (e.g., rotation of theelectrode pattern) in the same treatment region. Rotation, as used here,may refer to the rotation of the pattern of two or more electrodes,including (but not limited to) tissue penetrating electrodes, such asneedle electrodes. Rotation of the pattern of electrodes may be relativeto a target tissue region. In general, the rotated pattern may berotated by any amount of rotation (e.g., between 0.5 degrees to 359.5degrees, such as between 5 degrees and 355 degrees, between 10 degreesand 350 degrees, between 20 degrees and 340 degrees, between 30 degreesand 330 degrees, between 40 degrees and 320 degrees, approximately 90degrees, etc.). The rotation may be clockwise and/or counterclockwise.As described in detail below, rotation may be physical rotation of thepattern of electrodes (e.g., the applicator) relative to the tissue, orrotation by changing the active electrodes of an array of electrodes sothat the pattern of active electrodes is rotated relative to the targettissue. The pattern of electrodes may be rotated relative to a region oftissue (e.g., a target region of tissue) so that after rotation the sameregion of tissue is being modified. For example, the treatment tip maybe positioned on the same region of the tissue before and afterrotation.

For example, FIG. 17 illustrates a graph showing that electricalstimulation when the electrodes are rotated halfway through treatment(“rotation”) compared to lesions formed without rotating (“no rotation”)results in a reduction of the number of pulses by about 25% whilecreating a nearly identically-sized lesions (mean width of thelesions+/−SEM is shown on the left, mean length+/−SEM is shown on theright). In FIG. 17, tissue (e.g., pig skin) was treated with 134 pulsesduring treatment without rotating the electrodes (“No rotation”, n=4animals) using a pattern of electrodes on the end of an applicator tp. Asimilar region of tissue was treated with 100 pulses, but halfwaythrough the treatment (after 50 pulses), the treatment tip was rotatedat the same tissue region, in this example by 90 degrees. Tissue wasassessed after four days from the application of energy. Error bars oneach column in FIG. 17 are +/−SEM. Both the width and length of theresulting lesions were comparable. The treatment was otherwise identicalbetween the “rotation” and “no rotation” groups and was performed at thesame nanosecond pulse conditions with a 3 mm long trocar tip, using 300ns pulses. Tissue was assessed after four days from the application ofenergy.

Nearly identical results were seen with other tissue types as well. Forexample, FIG. 18 shows a similar trend for kidney tissue, using the sameexperimental protocol as in FIG. 17, and FIG. 19 shows the same trendwith liver tissue, again using the same experimental protocol as in FIG.17.

In FIG. 18, tissue was stimulated with 134 pulses during treatmentwithout rotating the electrodes (“No rotation”, n=2 animals), while only100 pulses were required to achieve a comparable lesion when theelectrode was rotated halfway through treatment by, in this example, 90degrees (“Rotation”, n=1 animals). Both the width and length of theselesions were comparable. All pulsing was performed at the samenanosecond pulse conditions with a 3 mm long trocar tip, using 300 nspulses. Tissue was assessed after four days from the application ofenergy. Error bars on each column in FIG. 18 are +/−SEM.

In FIG. 19, tissue was stimulated with 134 pulses during treatmentwithout rotating the electrodes (“No rotation”, n=2 animals), while only100 pulses were required to achieve a comparable lesion when theelectrode was rotated halfway through treatment by, in this example, 90degrees (“Rotation”, n=2 animals). Both the width and length of theselesions were comparable, as shown. All pulsing was performed at the samenanosecond pulse conditions with a 3 mm long trocar tip, using 300 nspulses. Tissue was assessed after four days from the application ofenergy. Error bars on each column in FIG. 19 are +/−SEM.

Thus, in general it may be beneficial when applying a treatment to atissue to rotate the pattern of electrodes applying energy to the tissuepartway through the treatment, since, in addition to other benefitsidentified above, it may require less energy, which may reduce any risksassociated with the procedure, and may speed tissue recovery.

FIG. 27 describes a general method of treating a tissue according tosome implementations of the present disclosure. The method may includecontacting a tissue with an applicator tip having a plurality ofelectrodes in a pattern of electrodes (step 2703). The tissue may be anytissue, and the electrodes on the applicator tip may be inserted intothe tissue, or, in some variations, placed against the tissue withoutpenetrating the tissue. A first portion of the treatment may then beapplied to a region of the tissue through a pattern of electrodes fromthe plurality of electrodes in a first orientation (step 2705). Beforeapplying the second portion of the treatment, in some implementation ofthe method the pattern of electrodes may be rotated by somepredetermined amount relative to the first orientation (e.g., between 5degrees and 175 degrees (or in some variations between 1 degree and 359degrees, etc.). In some variations the plurality of electrodes isrotated by 90 degrees. The rotation may be done manually, automatically,and/or semi-automatically. In some variations, the electrodes arerotated robotically, as discussed in greater detail below.Alternatively, rotation of the pattern of electrodes may be done byswitching the active electrodes in an array of electrodes. For example,the applicator tip may comprise an array of electrodes in which theplurality of electrodes is a first subset of active electrodes formingthe pattern of electrodes and a step of applying the second portion ofthe pulsed electric treatment comprises forming the pattern from asecond subset of active electrodes from the array of electrodes in whichthe pattern formed by the second subset is the same pattern but rotatedrelative to the first subset. Once the pattern is rotated relative tothe tissue, the second portion of the treatment may be applied to thesame region of the tissue in a second orientation (step 2707).

FIG. 28 describes an exemplary method in which the rotation of thepattern is achieved by changing a sub-set of active electrodes in anarray of electrodes that are used to apply the pulsed electrical energypart way through the treatment so that the pattern is rotated relativeto the initial subset of electrodes. For example, in FIG. 28, theapplicator (e.g., applicator tip) having an array of electrodes maycontact the tissue (step 2803). In some variations, the surface of thetissue may be contacted. In some variations, the electrodes maypenetrate the tissue. A pulsed electrical treatment (including, but notlimited to the application of nanosecond electrical pulses) may bedelivered by a sub-set of the electrodes in the array that are arrangedin a first pattern (step 2807). Partway through the treatment, andwithout removing the treatment tip from the tissue, switching to adifferent (e.g., second) sub-set of electrodes in the array arranged ina second pattern that is a rotated version of the first pattern (step2809). In other words, the second pattern may be exactly the same as thefirst pattern, except that its orientation is rotated relative to theorientation of the first pattern. The rotated version maybe rotated byany amount, which may be determined by the arrangement of the array ofelectrodes. For example, the pattern may be rotated by 25 degrees, by 45degrees, by 90 degrees, etc. Switching to the second (rotated) patternpartway through treatment may be switching midway (e.g., approximatelyhalfway) though the treatment, or switching after any appropriatefraction of the total treatment time (e.g., 30%, 40%, 50%, 60%, 70%,etc.). Switching to a different subset of electrodes may be accomplishedwithout moving the applicator tip or the array of electrodes, forexample, by simply activating different electrodes in the array ofelectrodes. Switching to a different subset of active electrodes may bedone automatically under control of a controller and/or a processor.

According to some implementations, a method of treating a tissue isprovided. The method comprises a treatment, such as a pulsed electricaltreatment, comprising a plurality of nanosecond electrical pulses havinga pulse duration of between 0.1 ns and 1000 ns, wherein the treatment isdivided into a first portion and a second portion (for example, whereinthe first portion is between 30% and 70% of the pulsed electricaltreatment). The method comprising contacting the tissue with anapplicator tip having a plurality of electrodes in a pattern ofelectrodes; applying the first portion of the treatment to a region ofthe tissue through the plurality of electrodes with the pattern ofelectrodes contacting the region of the tissue in a first orientation;removing the plurality of electrodes from the region of the tissue;rotating the applicator tip (for example, through a midline of theapplicator tip or a midline of the plurality of electrodes); re-applyingthe plurality of electrodes to the region of the tissue; and applyingthe second portion of the treatment to the same region of the tissuethrough the plurality of electrodes with the pattern of electrodescontacting the region of the tissue in a second orientation that isrotated relative to the first orientation.

It will be apparent that the number of steps of the methods that areutilized are not limited to those described above. Also, the methods donot require that all the described steps are present. Although themethodology described above as discrete steps, one or more steps may beadded, combined or even deleted, without departing from the intendedfunctionality of the embodiments of the disclosure. The steps can beperformed in a different order or have the steps shared between morethan one processor, for example. It will also be apparent that themethod described above may be performed in a partially or substantiallyautomated fashion, including performed using robotic systems.

Any energy delivery device or applicator, or applicator hand piece maybe used to apply the treatment and rotate the electrodes of such energydelivery device or applicator partway through the treatment. Therotation may be performed manually or automatically. In some variationsthe applicator is adapted to allow the pattern of electrodes at thedistal tip to be rotated at the target tissue partway through atreatment. For example, FIGS. 20A-20F illustrate one example of anapparatus that is configured to rotate the electrodes relative to thetissue partway through a treatment to the tissue. In FIGS. 20A-20B afirst configuration of an applicator is shown. FIG. 20A shows a sideview of a handle portion 2005 and a treatment tip 2007. A plurality ofelectrodes, shown as needle electrodes, are included on the distal endof the treatment tip, and are arranged in a pattern of two parallellines of needle electrodes 2009, shown in the front view of FIG. 20B,looking down onto the applicator tip.

The apparatus shown in FIG. 20A-20B is configured so that the pattern ofelectrodes may be rotated or turned as shown in FIG. 20C by arrows 2013either clockwise and/or counterclockwise partway through the treatment.As used herein, the term “partway” is not intended to require the exactone-half of the treatment but is intended to mean any desired orappropriate point during duration of the treatment. In FIG. 20C, theapplicator distal tip 2007 can be separately rotated, as shown by arrows2013. This rotation may be motorized, and may be controlled by a button,switch, or other controller, and may be automatically orsemi-automatically controlled. In FIG. 20D the tip is shown rotatingcounterclockwise (an approximately 45 degrees rotation is shown byexample). In some variations, as shown in FIGS. 20E and 20F, the patternof electrodes at the distal tip of the applicator may be rotated 90degrees (and/or other desired angles), as shown, and as is apparent inFIG. 20F.

In FIGS. 20A-20F, the pattern of electrodes on the treatment tip arerotated about a line of rotation 2025 along the treatment tip (in thelong axis of the applicator), and through a point of rotation in a planeof the pattern of electrodes. This line of rotation may, in somevariations, be referred to as a midline, as it may pass through thepoint of rotation 2025′ which may be in the middle (or center) of thepattern of electrodes in the plane of the electrodes. As shown in FIGS.20D and 20F, the pattern of electrodes rotates about this line ofrotation 2025.

Another variation, similar to the apparatus shown in FIGS. 15A-15D isshown in FIG. 21A. In this variation, the apparatus includes a handpiece2101 that may plug (via cord 2105) to a generator (not shown) forgenerating electrical stimulation, for example, the nano-pulsestimulation. The handpiece 2101 may be an energy delivery device in aform configured for the attachment to a movable arm of the roboticsystem, or in a form to be held by human operators. The distal endregion of the hand piece in this example is configured to becontrollably rotated during the application of energy (e.g., partwaythrough a treatment). In FIG. 21A, the hand piece may plug into agenerator and/or controller, which may also control the rotation. One ormore different tips (FIGS. 21B and 21C) may couple with the handpiece;the tips may include different patterns of electrodes for delivering theenergy to the tissue, as described above. In addition, in the variationshown in FIG. 21A, the handpiece may be configured so that theapplicator tip may controllably rotate, as shown by arrows 2104. Thisrotating region may include a turning joint and may be electricallydriven and controlled by user input and/or by an applicator controller(not shown). FIGS. 21B-21C illustrate exemplary electrode tips fortreating skin by delivering nano-pulse stimulation as described above.In FIG. 21A, the tip fits over the distal end of the headpiece 2101, andsnaps or locks in place while electrical contact is made with theprojecting (needle-like) electrodes 2111, 2111′. For example, the tipmay be mechanically secured (e.g., by snap-fit, friction fit, etc.) ontothe end of the handpiece. The handpiece may drive rotation of the distaltip.

In some variations, rather than a portion of the handpiece and/orapplicator tip move (e.g., rotate), the entire handpiece may be rotated.As mentioned this may be done manually. In some variations it may bebeneficial to rotate the handpiece robotically.

Alternatively or additionally, in some variations it may be desirable toavoid physical rotation of the electrodes. For example, in some of themanual implementation to avoid a possibility of targeting error and thepossibility of untreated or over-treated tissue, the methods fortreating tissue may be implemented without having to reposition or movethe electrodes while still achieving the benefits of the increasedtreatment volume and/or providing treatment in multiple directions. Suchimplementations are illustrated in FIGS. 22A-22B, 23A-23B, 24A-24B and25A-25C. In each of these examples, a front view looking down on theelectrodes is shown. Each of these tips includes an array of electrodesthat are either active (shown by shaded circles) or inactive (shown asopen circles). Active electrodes may be in contact with the tissue andconfigured to apply energy through the electrode into the tissue. Incontrast inactive electrodes may either be insulated from the tissue, soas not to electrically interact with the tissue, and/or they may bewithdrawn from the tissue, e.g., into the applicator tip. Thus, invariations in which the pattern of electrodes is rotated by switchingbetween sets of active electrodes, the applicator tip does not need torotate relative to the tissue but may remain on/in the tissue whilerotating the pattern of electrodes by changing which electrodes areactive or inactive partway through the treatment.

For example, FIG. 22A is an example of an applicator tip having a firstpattern of active electrodes, showing two parallel lines of threeelectrodes each. The applicator tip in this example includes a total of8 electrodes; in FIG. 22A, the two middle electrodes are inactive. FIG.22B illustrates rotation of the pattern of electrodes shown in FIG. 22Aby switching which electrodes are active; in this example, the sixvertical electrodes are active so that the two parallel lines of activeelectrodes resulting are perpendicular (e.g., rotated by 90 degrees)relative to the orientation of the active electrodes in FIG. 22A. Thefirst pattern of active electrodes may be rotated relative to tissuebeing treated by the electrodes by changing which electrodes of the tipof the applicator are active. The first pattern of active needleelectrodes is shown in the front view of FIG. 22A by the shaded circles2207; inactive needle electrodes are shown unshaded 2209. During use,partway through the treatment, the pattern of active electrodes may beswitched to that shown in FIG. 22B. This effectively rotates the patternof FIG. 22A by 90 degrees. In some variations, the electrodes may remainin the tissue. Alternatively or additionally, in some variations theinactive electrodes may be withdrawn from the tissue, includingwithdrawn axially into the applicator (e.g., into an insulated housingwithin the applicator). Thus, the applicator tip may remain in/on thetissue.

FIGS. 23A-23B illustrate a similar example, in which the initial patternof two parallel rows of five electrodes is rotated by inactivating someelectrodes and activating others to achieve the rotated configurationshown in FIG. 23B. In this example, the pattern of active electrodes isformed by two parallel lines of five electrodes. In FIG. 23A the firstpattern (two rows of five needle electrodes) is shown. In FIG. 23B, thesame first pattern is shown rotated by 90 degrees, for example. Thepattern of electrodes may be rotated by changing which electrodes areactive in the array of electrodes. Active needle electrodes are shown bythe shaded circles in the front views of FIGS. 23A and 23B; inactiveneedle electrodes are unshaded. Partway (e.g., halfway) through thetreatment, the pattern of active electrodes may be switched to from thepattern shown in FIG. 23A to the pattern shown in FIG. 23B to rotate theelectrodes relative to the tissue. In this example, the pattern isrotated by 90 degrees, while some of the electrodes remain in thetissue. Inactive electrodes may be withdrawn from the tissue, such as bybeing withdrawn axially into the applicator (e.g., into an insulatedhousing within the applicator). Thus, the applicator tip may remainin/on the tissue even during rotation partway through the treatment.

In any of the arrays of the treatment tips and/or arrays of electrodesdescribed herein, the plurality of electrodes may be arranged about aline of rotation along the treatment tip and/or applicator that passesthrough a point of rotation of the plurality of electrodes. This linemay pass through a center of the plurality of electrodes and may, insome variations, be referred to as a midline through the pattern ofelectrodes (“midline”) passing through a point of rotation at the centerof this pattern of electrodes (midpoint of the pattern of electrode inthe plane of the electrodes). An example of a line of rotation 2125through the electrodes is shown in the treatment tip of FIG. 21B. Thepattern formed by the electrodes (in some variations, the pattern ofactive electrodes) may be rotated about this line of rotation. In thisexample, the line of rotation is a midline trough both the array ofelectrodes and the treatment tip. In the frontal view of the electrodearray shown in FIGS. 22A-22B, the line of rotation is a midline thatpasses through a point of rotation 2225 that is in the center of thearray. The pattern of the active electrodes in FIG. 22A is shown rotatedabout this midline by 90 degrees in FIG. 22B. Similarly, in FIGS.23A-23B the line of rotation passes perpendicularly through the point2325 (point of rotation). The pattern of the electrodes shown in FIG.23A is rotated 90 degrees about this line of rotation, as shown in FIG.23B. The point of rotation may be the geometric center (central point)of the pattern of electrodes in the plane of the electrodes shown in thefrontal views. The point of rotation may be a center point determined byapproximating the distance from the centroid of each electrode to everyother electrode centroid in the array of electrodes. The point ofrotation is typically within the shape bounded by the array ofelectrodes (e.g., between them). In some variations the point ofrotation may be along one or more lines of symmetry in the plane of theelectrodes.

The electrode pattern shown in FIG. 24A shows a pair of electrodes thatmay be rotated by 90 degrees to achieve the pattern shown in FIG. 24B byswitching the active and inactive electrodes in the array of fourelectrodes shown. In this example, the pattern of active electrodes isformed by two perpendicular lines of two electrodes. In FIG. 24A thefirst pattern (a line of two needle electrodes) is shown by the shadedcircles, showing front views of the needle electrodes at the tip. InFIG. 24B, the same first pattern is shown rotated by 90 degrees. Thepattern of electrodes may be rotated while on a target tissue region(and without moving from the target tissue region) by changing whichelectrodes are active in the array of electrodes. Partway (e.g., midway)through the treatment, the pattern of active electrodes may be switchedto from the pattern shown in FIG. 24A to the pattern shown in FIG. 24Bto rotate the electrodes relative to the tissue.

As discussed above, any appropriate rotation of the electrode patterns,e.g., by switching of the active electrode patterns, may be used. Forexample, FIGS. 25A-25D illustrate rotation of counterclockwise 45degrees (between FIGS. 25A and 25B), rotation of 90 degrees (betweenFIGS. 25A and 25C) and rotation of clockwise 45 degrees (between FIGS.25A and 25D). The applicator tip (not shown) may remain in the sameposition relative to the tissue, while different sub-sets of electrodesin the applicator tip are made active or inactive, as shown.

As discussed above, the methods described herein are especially suitedfor use with a robotic system, and/or other automated and/orcomputer-implemented applications. For example, the apparatuses (e.g.,devices and systems) and methods described herein may be utilized invarious ablation procedures (e.g. radiation-based), dermatologicalprocedures (e.g., treating various dermatological conditions, such asskin cancers), etc.

FIG. 26 is a schematic perspective view of an example of a roboticsystem 100 that may be used to rotate the electrodes partway through atreatment. The system 100 may include a robotic arm 115 to which iscoupled an applicator 110, such as an energy delivery device, having anapplicator tip with a plurality of electrodes. Various motors and othermovement devices may be incorporated to enable fine movements of anoperating tip of the applicator 110 in multiple directions. The roboticsystem 100 may further include at least one (and preferably two forstereo vision, or more) image acquisition device 105 which may bemounted in a fixed position or coupled (directly or indirectly) to arobotic arm 115 or other controllable motion device. The operating tipof the applicator 110 may be positioned over a tissue (not shown).

The processor 125 of FIG. 26 may comprise, if applicable, an imageprocessor 130 for processing images obtained from the image acquisitiondevice 105. The image processor 130 may be a separate device or it maybe incorporated as a part of the processor 125. The processor 125 mayalso instruct the various movement devices of the robotic arm 115,including the applicator 110, and act, for example, through a controller135 as schematically shown in FIG. 26. The controller 135 may beoperatively coupled to the robotic arm and configured to control themotion of the robotic arm, including the motion based on the images ordata acquired by the image acquisition device. Alternatively, controller135 may be incorporated as a part of the processor 125, so that allprocessing and controls of all movements of all the tools, the roboticarm and any other moveable parts of the assembly, including those basedon the images or data acquired by the image acquisition device, areconcentrated in one place. The system 100 may further comprise a monitor140, mouse 150 and keyboard 160. In addition, the system 100 maycomprise other tools, devices and components useful in treating atissue, including with a pulsed electrical treatment. The system mayfurther include an interface (not shown) adapted to receive an imagedata, various parts of the system allow an operator to monitorconditions and provide instructions, as needed. The processor 125 mayinteract with the imaging device 105 via the interface. The interfacemay include hardware ports, cables, leads, and other data transmissionmeans, or it may comprise a computer program.

Some non-limiting examples of an image acquisition device 105 shown inFIG. 26 include one or more cameras, such as any commercially availablecameras. The image acquisition or imaging device may be held, forexample, by a robotic arm, or by any other mechanism or means. Variousimage acquisition devices or a combination of several devices could beused with any of the embodiments of the systems and methods describedherein. The image acquisition device 105 may comprise a device thattakes still images, it can also comprise a device capable of real timeimaging (e.g., webcam capable of continuously streaming real timeinformation), and/or it could also have a video recording capability(such as a camcorder, or smart phone or other mobile device). Whilestereo or multi-view imaging devices are very useful in the presentdisclosure, it is not necessary to employ such geometries orconfigurations, and the present disclosure is not so limited. The imageacquisition device may be coupled to a processing system 125, shownincorporated in the image processor 130 in FIG. 26, to control theimaging operation and process image data.

Typically, the processor 125 operates as a data processing device, forexample, it may be incorporated into a computer. The processor 125 mayinclude a central processing unit or parallel processor, andinput/output interface, a memory with a program, wherein all thecomponents may be connected by a bus. Further, the computer may includean input device, a display, and may also include one or more secondarystorage devices. The bus may be internal to the computer and may includean adapter for receiving a keyboard or input device or may includeexternal connections.

The processor 125 may execute a program that may be configured toinclude predetermined operations. The processor may access the memory inwhich may be stored at least one sequence of code instructionscomprising the program for performing predetermined operations. Thememory and the program may be located within the computer or may belocated external thereto. By way of example, and not limitation, asuitable image processor 130 may be a digital processing system whichincludes one or more processors or other type of device. For example, aprocessor and/or an image processor may be a controller or any type ofpersonal computer (“PC”). Alternatively, the processor may comprise anApplication Specific Integrated Circuit (ASIC) or Field ProgrammableGate Array (FPGA). It will be understood by those of ordinary skill inthe art that the processor and/or the image processor for use with thepresent disclosure is programmed and configured to perform various knownimage processing techniques, for example, segmentation, edge detection,object recognition and selection. These techniques are generally knownand do not need to be separately described here. The methods describedherein may be implemented on various general or specific purposecomputing systems. In certain embodiments, the methods of the presentapplication may be implemented on a specifically configured personalcomputer or workstation. In other embodiments, the methods may beimplemented on a general-purpose workstation, including one connected toa network. Alternatively or additionally, the methods of the disclosuremay be, at least partially, implemented on a card for a network deviceor a general-purpose computing device. The processor/image processor mayalso include memory, storage devices, and other components generallyknown in the art and, therefore, they do not need to be described indetail here. The image processor could be used in conjunction withvarious manual, partially automated and fully automated (includingrobotic) systems and devices.

The imaging display device 140 may comprise a high resolution computermonitor which may optionally be a touch screen. The imaging display mayallow images, such as video or still images, to be readable and forfollicular units, and parts thereof, to be visualized. Alternatively,the imaging display device 140 can be other touch sensitive devices,including tablet, pocket PC, and other plasma screens. The touch screenmay be used to modify the parameters of the hair transplantationprocedure, directly through the image display device.

Methods, and apparatuses of the present disclosure may be carried out byproviding a modification interface, or user modification interface,including touch screen, clickable icons, selection buttons in a menu,dialog box, or a roll-down window of an interface that may be providedto feed into the computer. According to another embodiment, the imagingdisplay device 140 may display the selection window and a stylus orkeyboard for entering a selection, for example, directly on the displayitself. According to one embodiment, commands may be input via themodification interface through a programmable stylus, keyboard, mouse,speech processing system, laser pointer, touch screen, tablet computer,personal digital assistant (PDA), a remote input device (such as apendant), or other input mechanism. The remote input device may includeclickable icons, selection buttons, dialog boxes, or roll-down windowswhich are the same as or similar to those found on the user modificationinterface, providing a convenient way for the user to control commonuser interface functions from their position at the patient's side.Alternatively, the remote input device may only accommodate, forexample, a subset of such modification controls, making for a morecompact pendant. In yet another embodiment, the remote input device maybe configured to accommodate additional modification controls. Moreover,either the remote input device or any other input mechanism may haveicons which allow the user to control the robotic arm, allowing the usermove the robotic arm away from the patient, or incorporate a STOPbutton, enabling the user to terminate operation of the robotic arm orthe applicator in the event of an emergency. Alternatively, themodification interface may comprise a dedicated piece of hardware. Insome embodiments the selections or adjustment made through themodification interface may be executed by code instructions that may beexecuted on the computer processor.

Embodiments of the methods of the present disclosure may be implementedusing computer software, firmware or hardware. Various programminglanguages and operating systems may be used to implement the presentdisclosure. The program that runs the method and system may include aseparate program code including a set of instructions for performing adesired operation or may include a plurality of modules that performsuch sub-operations of an operation or may be part of a single module ofa larger program providing the operation. The modular constructionfacilitates adding, deleting, updating and/or amending the modulestherein and/or features within the modules.

In some embodiments, a user may select a particular method or embodimentof this application, and the processor will run a program or algorithmassociated with the selected method. In certain embodiments, varioustypes of position sensors may be used. For example, in certainembodiment, a non-optical encoder may be used where a voltage level orpolarity may be adjusted as a function of encoder signal feedback toachieve a desired angle, speed, or force.

The processor for use in the present disclosure may comprise anysuitable device programmed and configured to perform various methodsdescribed herein. In some embodiments modification may be accomplishedthrough the modification interface. For example, the processor for usein the present disclosure may be a processor comprising a set ofinstructions for executing operations, the set of instructions includinginstructions for applying treatment via the applicator. The user mayinput how much of the treatment to perform before rotating the pattern,what energy to apply before and/or after rotating, the duration of thetreatment, the location of the treatment, the tissue impedance changebefore each rotation, and any other appropriate parameter. A system foruse according to the disclosures described herein may comprise inaddition to a processor an image acquisition device. Thus, any of theseapparatuses may include a user input device, the user input deviceconfigured to allow a user to interactively modify any of theseparameters. In other embodiments, the processor is configured toautomatically modify these parameters (e.g., input how much of thetreatment to perform before rotating the pattern, what energy to apply,the duration of the treatment, the location of the treatment, etc.).

Certain embodiments relate to a machine-readable medium (e.g., computerreadable media) or computer program products that include programinstructions and/or data (including data structures) for performingvarious computer-implemented operations. A machine-readable medium maybe used to store software and data which causes the system to performmethods of the present disclosure. The above-mentioned machine-readablemedium may include any suitable medium capable of storing andtransmitting information in a form accessible by processing device, forexample, a computer. Some examples of the machine-readable mediuminclude, but not limited to, magnetic disc storage such as hard disks,floppy disks, magnetic tapes. It may also include a flash memory device,optical storage, random access memory, etc. The data and programinstructions may also be embodied on a carrier wave or other transportmedium. Examples of program instructions include both machine code, suchas produced by a compiler, and files containing higher level code thatmay be executed using an interpreter.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to perform or control performing of any of the steps,including but not limited to: displaying, communicating with the user,analyzing, modifying parameters (including timing, frequency, intensity,etc.), determining, alerting, or the like. In some exemplary embodimentshardware may be used in combination with software instructions toimplement the present disclosure.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. For example, asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present apparatuses andmethods.

The terms “comprises” and/or “comprising,” when used in thisspecification (including the claims), specify the presence of statedfeatures, steps, operations, elements, and/or components, but do notpreclude the presence or addition of one or more other features, steps,operations, elements, components, and/or groups thereof. Unless thecontext requires otherwise, “comprise”, and variations such as“comprises” and “comprising,” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

Any of the apparatuses and methods described herein may include all or asub-set of the components and/or steps, and these components or stepsmay be either non-exclusive (e.g., may include additional componentsand/or steps) or in some variations may be exclusive, and therefore maybe expressed as “consisting of” or alternatively “consisting essentiallyof” the various components, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the disclosure as described by the claims. Forexample, the order in which various described method steps are performedmay often be changed in alternative embodiments, and in otheralternative embodiments one or more method steps may be skippedaltogether. Optional features of various device and system embodimentsmay be included in some embodiments and not in others. Therefore, theforegoing description is provided primarily for exemplary purposes andshould not be interpreted to limit the scope of the apparatuses andmethods as it is set forth in the claims.

Various embodiments may be referred to herein individually orcollectively by the term “invention” merely for convenience and withoutintending to voluntarily limit the scope of this application to anysingle invention or inventive concept, if more than one is, in fact,disclosed. Thus, although specific embodiments have been illustrated anddescribed herein, any arrangement calculated to achieve the same purposemay be substituted for the specific embodiments shown. This disclosureis intended to cover any and all adaptations or variations of variousembodiments. Combinations of the above embodiments, and otherembodiments not specifically described herein, will be apparent to thoseof skill in the art upon reviewing the above description.

What is claimed is:
 1. A method of treating a tissue with a pulsedelectrical treatment comprising a plurality of electrical pulses,wherein the pulsed electrical treatment is divided into at least a firstportion and a second portion, the method comprising: contacting thetissue with an applicator tip having a plurality of electrodes, whereinthe applicator tip is configured to removably couple to a handpiece;applying the first portion of the pulsed electrical treatment to aregion of the tissue in a pattern of electrodes from the plurality ofelectrodes, the pattern of electrodes contacting the region of thetissue in a first orientation; rotating the pattern of electrodes of theapplicator tip by rotating at least a portion of the applicator tiprelative to the handpiece to which the applicator tip is coupled; andapplying the second portion of the pulsed electrical treatment to theregion of the tissue in the pattern of electrodes contacting the regionof the tissue in the second orientation that is rotated relative to thefirst orientation.
 2. The method of claim 1, wherein the secondorientation is rotated between 40 degrees and 100 degrees relative tothe first orientation.
 3. The method of claim 1, wherein the secondportion of the pulsed electrical treatment is applied through the sameelectrodes of the plurality of electrodes that the first portion of thepulsed electrical treatment is applied through.
 4. The method of claim1, wherein the second orientation is rotated about a midline through theplurality of electrodes.
 5. The method of claim 1, wherein the pulseelectrical treatment comprises a plurality of electrical pulses having apulse duration of between 0.1 ns and 1000 ns.
 6. The method of claim 1,wherein the first portion of the treatment is between 30% and 70% of thepulsed electrical treatment.
 7. The method of claim 1, furthercomprising removing the plurality of electrodes from the region of thetissue before rotating the at least the portion of the applicator tipand re-applying the plurality of electrodes to the region of the tissuebefore applying the second portion of the pulsed electrical treatment.8. The method of claim 7, wherein removing, rotating and reapplying areperformed by a robotic system.
 9. The method of claim 1, whereinapplying the pulsed electrical treatment comprises applying anon-thermal treatment that does not disrupt cell membranes within thetissue.
 10. The method of claim 1, wherein applying the first and secondportions of the pulsed electrical treatment comprises applying for lessthan 5 minutes.
 11. The method of claim 1, wherein contacting the tissuecomprises contacting a skin tissue.
 12. The method of claim 11, whereinthe skin tissue comprises one or more of: seborrheic keratosis, keloids,molluscum contagiosum, sebaceous hyperplasia, acrocordon, syringoma,psoriasis, papilloma, congenital capillary malformation (port-winestain), human papilloma virus (HPV), melanoma, melasma, actinickeratoses, dermatosis papulosa nigra, angiofibroma, skin tumors, agedskin, wrinkled skin, and warts.
 13. The method of claim 1, whereinapplying the pulsed electrical treatment increases a marker ofinflammation within the region of the tissue by less than 15%, whereinthe marker of inflammation is one or of more of: fibroblast density,leukocyte density, Interleukin-6, Interleukin-8, Interleukin-18, Tumornecrosis factor-alpha, and C-reactive protein.
 14. The method of claim1, wherein the applying the first portion of the pulsed electricaltreatment and the applying the second portion of the electricaltreatment each comprises applying electrical pulses to the region tode-nucleate cells within the region without provoking a substantialinflammatory response, so that after the treatment the tissue forms anecrotic crust over the region so that when the necrotic crust isremoved new tissue is exposed.
 15. The method of claim 1, whereinrotating the pattern of electrodes comprises rotating the pattern ofelectrodes when a user operates a control to drive rotation of the atleast the portion of the applicator tip.
 16. The method of claim 1,wherein rotating the pattern of electrodes comprises automaticallyrotating the pattern of electrodes under the control of a controller todrive rotation of the at least the portion of the applicator tip.
 17. Amethod of treating a tissue with a pulsed electrical treatment, whereinthe pulsed electrical treatment is divided into a first portion and asecond portion, the method comprising: contacting a region of the tissuewith an applicator tip having a plurality of electrodes in a pattern ofelectrodes, wherein the applicator tip is configured to removably coupleto a handpiece; applying the first portion of the pulsed electricaltreatment to the region of the tissue through the plurality ofelectrodes with the pattern of electrodes contacting the region of thetissue in a first orientation; removing the plurality of electrodes fromthe region of the tissue; rotating the pattern of electrodes of theapplicator tip by rotating the applicator tip relative to the handpieceto which the applicator tip is coupled; re-applying the plurality ofelectrodes to the region of the tissue with the pattern of electrodescontacting the region of the tissue in a second orientation that isrotated relative to the first orientation; and applying the secondportion of the pulsed electrical treatment to the region of the tissuethrough the plurality of electrodes.
 18. The method of claim 17, whereinthe pulse electrical treatment comprises a plurality of electricalpulses having a pulse duration of between 0.1 ns and 1000 ns.
 19. Amethod of treating a tissue with a pulsed electrical treatmentcomprising a plurality of nanosecond electrical pulses having a pulseduration of between 0.1 ns and 1000 ns, wherein the pulsed electricaltreatment is divided into a first portion and a second portion, themethod comprising: contacting the tissue with an applicator tip having aplurality of electrodes, wherein the applicator tip is configured toremovably couple to a handpiece; applying the first portion of thepulsed electrical treatment to a region of the tissue in a pattern ofelectrodes from the plurality of electrodes, the pattern of electrodescontacting the region of the tissue in a first orientation; rotating thepattern of electrodes of the applicator tip about a midline of theapplicator tip passing through the plurality of electrodes, by selectinga subset of the plurality of electrodes forming the same pattern ofelectrodes in a second orientation that is radially offset from thefirst orientation; and applying the second portion of the pulsedelectrical treatment to the region of the tissue in the pattern ofelectrodes contacting the region of the tissue in the second orientationthat is rotated relative to the first orientation.
 20. The method ofclaim 19, further comprising rotating the pattern of electrodes on theapplicator tip without removing the applicator tip from the tissue. 21.The method of claim 19, wherein contacting the tissue comprisescontacting the tissue with the applicator tip wherein the applicator tipcomprises an array of electrodes in which the plurality of electrodes isa first subset of active electrodes forming the pattern of electrodes;and wherein applying the second portion of the pulsed electricaltreatment comprises forming the pattern from a second subset of activeelectrodes from the array of electrodes in which the pattern formed bythe second subset is rotated relative to the first subset.
 22. Themethod of claim 19, wherein the method is at least partially automated,the method comprising automatically rotating the pattern of electrodesfrom the first orientation to the second orientation.
 23. The method ofclaim 19, wherein a degree of rotation from the first orientation to thesecond orientation is user selected through a user interface orautomatically directed by a controller.
 24. The method of claim 19,further comprising selecting or allowing selection of one or more of thefollowing: amount of energy to apply during the applying the firstand/or the second portion of the pulsed electrical treatment, apercentage of one or both of the first portion and the second portion ofthe pulsed electrical treatment, a degree or amount of rotation of thepattern of electrodes, a duration of the pulsed electrical treatment, ora tissue impedance change before each rotation.
 25. The method of claim19, wherein any one or more of the steps of the contacting, applying thefirst portion, rotating, or applying the second portion is performedunder control of a processor.